H3.3 CTL peptides and uses thereof

ABSTRACT

Peptides that generate an immune response to glioma-related H3.3 proteins and methods of their use are provided.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present patent application is a continuation of U.S. patentapplication Ser. No. 15/613,837, filed Jun. 5, 2017, which is acontinuation-in-part of International Application No. PCT/US16/30849,filed May 4, 2016, which claims benefit of priority to U.S. ProvisionalPatent Application No. 62/157,362, filed May 5, 2015, and U.S.Provisional Patent Application No. 62/212,508, filed Aug. 31, 2015, thedisclosures of each of which are incorporated by reference in theirentireties.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under grant no. R21NS083171 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

INCORPORATION BY REFERENCE

This application contains a Sequence Listing in computer readable form(filename: 048536-638C01US_SequenceListing_ST25.txt; 12,324 bytes—ASCIItext file; created on Jan. 6, 2020), which is incorporated herein byreference in its entirety and forms part of the disclosure.

BACKGROUND OF THE INVENTION

Malignant gliomas, including glioblastomas (GBM), diffuse intrinsicpontine gliomas (DIPG), ependymomas, astrocytomas, oligodendrogliomas,brainstem glioma, thalamic gliomas, spinal cord gliomas, and optic nerveglioma, are lethal brain tumors in both adults and children. Recentgenetic studies have revealed that malignant gliomas in children andyoung adults often show recurrent missense mutations in H3F3A, whichencodes the replication-independent histone 3 variant H3.3. See, e.g.,Lewis et al., Science Vol. 340 no. 6134 pp. 857-861 (2013).Approximately 30% of overall GBM and 70% of DIPG cases harbor theamino-acid substitution from lysine (K) to methionine (M) at theposition 27 of H3.3 (K27M mutation, hereafter), which is universallyassociated with shorter survival in DIPG patients compared with patientswith non-mutated H3.3. The adaptive immune system, such as T lymphocytes(T cells hereafter), are normally tolerant to normal self-proteins, butcan recognize mutated amino-acids as non-self. Hence cancer-specificmutations can be suitable targets of cancer immunotherapy, such ascancer vaccines and adoptive T cell transfer therapy.

BRIEF SUMMARY OF THE INVENTION

In some embodiments, an isolated peptide consisting of less than 100,75, 50, or 30 amino acids is provided, wherein the peptide comprises(R/A)MSAP(S/A)TGGV (SEQ ID NO:1 In some embodiments, the peptideconsists of 10-14 amino acids. In some embodiments, the peptide consistsof (R/A)MSAP(S/A)TGGV (SEQ ID NO:1). In some embodiments, the R in SEQID NO:1 is citrullinated. In some embodiments, the R in SEQ ID NO:1 isnot citrullinated.

Also provided is a fluorochrome-conjugated peptide-majorhistocompatibility complex (pMHC) multimer, wherein the peptide is asdescribed above or elsewhere herein.

Also provided is a nucleic acid, optionally isolated or purified,encoding the peptide as described above or elsewhere herein. In someembodiments, the nucleic acid is a plasmid or a viral vector. In someembodiments, the vector is capable of delivering the nucleic acid intoan antigen presenting cell (APC).

Also provided is a cell comprising a heterologous peptide consisting of10-12 or 10-14 (e.g., 10, 11, 12, 13, or 14) amino acids, wherein thepeptide comprises (R/A)MSAP(S/A)TGGV (SEQ ID NO:1). In some embodiments,the peptide consists of (R/A)MSAP(S/A)TGGV (SEQ ID NO:1). In someembodiments, the R in SEQ ID NO:1 is citrullinated. In some embodiments,the R in SEQ ID NO:1 is not citrullinated. In some embodiments, the cellis an antigen presenting cell (APC). In some embodiments, the cell is abacterial cell. In some embodiments, the bacterial cell is E. coli orListeria monocytogenes. In some embodiments, the APC presents theheterologous peptide on the surface of the cell. In some embodiments,the APC comprises a heterologous expression cassette comprising apromoter operably linked to a polynucleotide encoding the peptide.

Also provided is a method of inducing an immune response in a humanindividual. In some embodiments, the method comprises administering asufficient amount of the APC as described above or elsewhere herein tothe human individual, thereby inducing an immune response in the humanindividual to replication-independent histone 3 variant H3.3 or H3.1. Insome embodiments, the APC is from the individual (autologous). In someembodiments, the immune response is a cytotoxic T-cell response. In someembodiments, the human individual has a glioma. In some embodiments, theindividual carries an HLA-*0201, HLA-*0202, HLA-*0203, HLA-*0204,HLA-*0205, HLA-*0206, HLA-*0207, or HLA-*0211 allele.

Also provided is a composition for stimulating an immune response toreplication-independent histone 3 variant H3.3, the compositioncomprises a peptide consisting of 10-12 or 10-14 (e.g., 10, 11, 12, 13,or 14) amino acids, wherein the peptide comprises (R/A)MSAP(S/A)TGGV(SEQ ID NO:1). In some embodiments, the composition comprises a peptideconsisting of (R/A)MSAP(S/A)TGGV (SEQ ID NO:1). In some embodiments, thecomposition further comprises an adjuvant. In some embodiments, the R inSEQ ID NO:1 is citrullinated. In some embodiments, the R in SEQ ID NO:1is not citrullinated.

Also provided is a method of inducing an immune response in a humanindividual, the method comprising administering a sufficient amount ofthe composition as described above or elsewhere herein to the humanindividual, thereby inducing an immune response in the human individualto histone 3 variant H3.3 or H3.1. In some embodiments, the compositioncomprises an adjuvant selected from the group consisting of polyICLC andBacillus Calmette-Guérin (BCG) vaccine. In some embodiments, the immuneresponse is a cytotoxic T-cell response. In some embodiments, the humanindividual has a glioma. In some embodiments, the individual carries anHLA-*0201, HLA-*0202, HLA-*0203, HLA-*0204, HLA-*0205, HLA-*0206,HLA-*0207, or HLA-*0211 allele.

Also provided is an antibody that specifically binds to RMSAPSTGGV (SEQID NO:2) and does not bind to RKSAPSTGGV (SEQ ID NO:3). In someembodiments, the antibody is linked to a heterologous detectable label.In some embodiments, the label is fluorescent.

Also provided is a T-cell expressing one or more polypeptides comprisinga T-cell receptor (TCR), or a peptide/MHC complex-binding fragmentthereof or a peptide/HLA complex-binding fragment thereof, that bindsthe peptide as described above or elsewhere herein in a peptide/MHCcomplex or peptide/HLA complex. In some embodiments, the TCR isheterologous to the T-cell. In some embodiments, expression of the TCRis under the control of a heterologous promoter (e.g., as transgenes).In some embodiments, the TCR comprises one or more of the CDRs as listedin SEQ ID NOs:12-17, optionally in a heterologous framework region. TheTCR will in general be formed of an alpha and a beta chain (two separatepolypeptides). In some embodiments, the TCR comprises SEQ ID NO:8, SEQID NO:10, or both, either as a fusion protein or as separate proteins.In some embodiments, the TCR comprises complementarity determiningregions (CDRs) as listed in SEQ ID NOs:12-17 (i.e., alpha chain CDR 1,2, and 3 being SEQ ID NOs; 12, 13, and 14, respectively, and beta chainCDRs 1, 2, and 3 being SEQ ID NOs; 15, 16, and 17, respectively). Insome embodiments, the glioma cell is a glioblastoma (GBM) cell. In someembodiments, the glioma cell is a diffuse intrinsic pontine glioma(DIPG) cell. In some embodiments, the glioma cell is a ependymoma,astrocytoma, oligodendroglioma, brainstem glioma, thalamic glioma,spinal cord glioma, or optic nerve glioma. In some embodiments, the TCRcomprises a first polypeptide comprising a TCR alpha chain and a secondpolypeptide comprising a TCR beta chain.

Also provided is an isolated nucleic acid encoding an alpha chain, abeta chain, or both an alpha chain and a beta chain of the T-cellreceptor (TCR) or a peptide/MHC complex-binding fragment thereof or apeptide/HLA complex-binding fragment thereof as described above orelsewhere herein. In some embodiments, the TCR comprises alpha chaincomplementarity determining region (CDR) 1, 2, and 3 being SEQ ID NOs;12, 13, and 14, respectively, and beta chain CDRs 1, 2, and 3 being SEQID NOs; 15, 16, and 17, respectively.

Also provided is an expression cassette comprising a promoter operablylinked to the nucleic acid as described above or elsewhere herein. Insome embodiments, the promoter is heterologous to the nucleic acid.

Also provided is a method of targeting T-cells to cells (e.g., gliomacells) expressing histone 3 variant H3.3 or H3.1 in an individual inneed thereof. In some embodiments, the method comprises, administeringto the individual a T-cell expressing a polypeptide comprising a T-cellreceptor (TCR), or a peptide/MHC complex binding fragment thereof or apeptide/HLA complex binding fragment thereof, that binds(R/A)MSAP(S/A)TGGV (SEQ ID NO:1) in a peptide/MHC complex or peptide/HLAcomplex, thereby targeting the T-cell to glioma cells expressing histone3 variant H3.3 or H3.1. In some embodiments, the TCR is heterologous tothe T-cell. In some embodiments, expression of the TCR is under thecontrol of a heterologous promoter. In some embodiments, the TCRcomprises complementarity determining regions (CDRs) as listed in SEQ IDNOs:12-17 (i.e., alpha chain CDR 1, 2, and 3 being SEQ ID NOs; 12, 13,and 14, respectively, and beta chain CDRs 1, 2, and 3 being SEQ ID NOs;15, 16, and 17, respectively). In some embodiments, the glioma cell is aglioblastoma (GBM) cell. In some embodiments, the glioma cell is adiffuse intrinsic pontine glioma (DIPG) cell. In some embodiments, theglioma cell is a ependymoma, astrocytoma, oligodendroglioma, brainstemglioma, thalamic glioma, spinal cord glioma, or optic nerve glioma.

Also provided is a method of enriching for T-cells expressing a TCR thatbinds the peptide as described above or elsewhere herein. In someembodiments, the method comprises generating a starting culture ofT-cells expressing TCRs; culturing the T-cells in the presence of thepeptide to generate an enriched culture of T-cells, wherein the enrichedculture is enriched for T-cells expressing TCRs that bind the peptidecompared to the starting culture. In some embodiments, the culturingcomprises culturing the T-cells in the presence of IL-2, IL-4, IL-7,IL-15, or combinations thereof. In some embodiments, the method furthercomprises sorting cells in the enriched culture for T-cells that bindthe peptide in a peptide/MHC complex to form a further enrichedpopulation of T-cells expressing TCRs that bind the peptide/MHC complex.In some embodiments, the sorting comprises contacting cells in theenriched culture with a fluorochrome-conjugated peptide-majorhistocompatibility complex (pMHC) multimer.

Definitions

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term encompasses nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms encompass amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as naturally occurring amino acid polymersand non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. The term “aminoacid analogs” refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an a carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. The term “amino acid mimetics”refers to chemical compounds that have a structure that is differentfrom the general chemical structure of an amino acid, but that functionsin a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and UGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

For amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins whichcan be made detectable, e.g., by incorporating a radiolabel into thepeptide or used to detect antibodies specifically reactive with thepeptide.

An antibody can consist of one or more polypeptides substantiallyencoded by immunoglobulin genes or fragments of immunoglobulin genes.The recognized immunoglobulin genes include the kappa, lambda, alpha,gamma, delta, epsilon and mu constant region genes, as well as myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively. An “antibody” functions as abinding protein and is structurally defined as comprising an amino acidsequence from or derived from the framework region of animmunoglobulin-encoding gene of a vertebrate animal.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

The term “antibody” as used herein encompasses antibody fragments thatretain antigen-binding specificity. For example, there are a number ofwell characterized antibody fragments. Thus, for example, pepsin digestsan antibody C-terminal to the disulfide linkages in the hinge region toproduce F(ab)′2, a dimer of Fab which itself is a light chain joined toVH-CH1 by a disulfide bond. The F(ab)′2 may be reduced under mildconditions to break the disulfide linkage in the hinge region therebyconverting the (Fab′)2 dimer into an Fab′ monomer. The Fab′ monomer isessentially an Fab with part of the hinge region (see, e.g., FundamentalImmunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a moredetailed description of other antibody fragments). While variousantibody fragments are defined in terms of the digestion of an intactantibody, one of skill will appreciate that fragments can be synthesizedde novo either chemically or by utilizing recombinant DNA methodology.Thus, the term antibody, as used herein also includes antibody fragmentseither produced by the modification or digestion of whole antibodies orsynthesized using recombinant DNA methodologies.

Antibodies can include V_(H)-V_(L) dimers, including single chainantibodies (antibodies that exist as a single polypeptide chain), suchas single chain Fv antibodies (sFv or scFv) in which a variable heavyand a variable light region are joined together (directly or through apeptide linker) to form a continuous polypeptide. The single chain Fvantibody is a covalently linked V_(H)-V_(L) which may be expressed froma nucleic acid including V_(H)- and V_(L)-encoding sequences eitherjoined directly or joined by a peptide-encoding linker (e.g., Huston, etal. Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). While the V_(H) andV_(L) are connected to each as a single polypeptide chain, the V_(H) andV_(L) domains associate non-covalently. Alternatively, the antibody canbe another fragment. Other fragments can also be generated, e.g., usingrecombinant techniques, as soluble proteins or as fragments obtainedfrom display methods. Antibodies can also include diantibodies andminiantibodies. Antibodies of the invention also include heavy chaindimers, such as antibodies from camelids. Thus, in some embodiments anantibody is dimeric. In other embodiments, the antibody may be in amonomeric form that has an active isotype. In some embodiments theantibody is in a multivalent form, e.g., a trivalent or tetravalentform.

As used herein, “complementarity-determining region (CDR)” refers to thethree hypervariable regions in each chain that interrupt the four“framework” regions established by the light and heavy chain variableregions. The CDRs are primarily responsible for binding to an epitope ofan antigen. The CDRs of each chain are typically referred to as CDR1,CDR2, and CDR3, numbered sequentially starting from the N-terminus, andare also typically identified by the chain in which the particular CDRis located. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found.

As used herein, “V-region” refers to an antibody variable region domaincomprising the segments of Framework 1, CDR1, Framework 2, CDR2, andFramework3, including CDR3 and Framework 4, which segments are added tothe V-segment as a consequence of rearrangement of the heavy chain andlight chain V-region genes during B-cell differentiation.

The sequences of the framework regions of different light or heavychains are relatively conserved within a species. The framework regionof an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three dimensional space.

The amino acid sequences of the CDRs and framework regions can bedetermined using various well known definitions in the art, e.g., Kabat,Chothia, international ImMunoGeneTics database (IMGT), and AbM (see,e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structuresfor the hypervariable regions of immunoglobulins. J. Mol. Biol. 196,901-917; Chothia C. et al., 1989, Conformations of immunoglobulinhypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992,structural repertoire of the human VH segments J. Mol. Biol. 227,799-817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)). Definitions ofantigen combining sites are also described in the following: Ruiz etal., IMGT, the international ImMunoGeneTics database. Nucleic AcidsRes., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the internationalImMunoGeneTics database. Nucleic Acids Res. January 1; 29(1):207-9(2001); MacCallum et al, Antibody-antigen interactions: Contact analysisand binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); andMartin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin,et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al,Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M. J. E.(ed.), Protein Structure Prediction. Oxford University Press, Oxford,141-172 1996).

“Epitope” or “antigenic determinant” refers to a site on an antigen towhich an antibody binds. Epitopes can be formed both from contiguousamino acids or noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed (1996).

As used herein, “chimeric antibody” refers to an immunoglobulin moleculein which (a) the constant region, or a portion thereof, is altered,replaced or exchanged so that the antigen binding site (variable region)is linked to a constant region of a different or altered class, effectorfunction and/or species, or an entirely different molecule which confersnew properties to the chimeric antibody, e.g., an enzyme, toxin,hormone, growth factor, drug, etc.; or (b) the variable region, or aportion thereof, is altered, replaced or exchanged with a variableregion, or portion thereof, having a different or altered antigenspecificity; or with corresponding sequences from another species orfrom another antibody class or subclass.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, often in a heterogeneous population ofproteins and other biologics such as a mixture of cells or a celllysate. Thus, under designated immunoassay conditions, the specifiedantibodies bind to a particular protein (e.g., SEQ ID NO:2) at least twotimes the background and more typically more than 10 to 100 timesbackground. Specific binding to an antibody under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies can be selectedto obtain only those polyclonal antibodies that are specificallyimmunoreactive with the selected antigen and not with other proteins.This selection may be achieved by subtracting out antibodies thatcross-react with other molecules. A variety of immunoassay formats maybe used to select antibodies specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual(1998) for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity).

“Antigen presenting cells”, or “APCs” are cells that cells that mediatethe cellular immune response by processing and presenting antigens tothe T-cell receptor and include Langerhans cells, veiled cells ofafferent lymphatics, dendritic cells and interdigitating cells oflymphoid organs. APCs include mononuclear cells such as lymphocytes andmacrophages.

A polynucleotide or polypeptide sequence is “heterologous” to anorganism or a second polynucleotide sequence if it originates from aforeign species, or, if from the same species, is modified from itsoriginal form. For example, when a heterologous promoter is said to beoperably linked to a coding sequence, it means that the coding sequenceis derived from one species whereas the promoter sequence is derivedfrom another, different species; or, if both are derived from the samespecies, the coding sequence is not naturally associated with thepromoter (e.g., the promoter is a genetically engineered promoter orpromoter fragment not found naturally associated with the codingsequence).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Identification of a HLA-A*0201-restricted epitope in H3.3 withthe K27M mutation. A. HLA-A201 binding ability of H3.3-derived peptideswas analyzed by T2 cell A2-binding assay. Cap1-6D is an altered peptideligand, which has been derived from an epitope in human carcinoembryonicAg, CEA605-613 and was used as positive control. B. H3.3 tetramerstaining analysis of the CD8+ CTLs generated with the H3.3.K27M (26-35)after 1st antigen stimulation (left) and weekly re-stimulations (right).

FIGS. 2A-C. HLA-A*0201+ donor-derived CTLs specifically recognizeHLA-A*0201+ K27M+ glioma cells in an HLA-class I-dependent manner.Peripheral blood mononuclear cells from an HLA-A*0201+ donor werestimulated in vitro with the H3.3.K27M peptide and evaluated for theirreactivity against: (1A) HLA-A*0201/H3.3.K27M-specific tetramer andanti-CD8 mAb, and (1B) T2 cells pulsed with the mutant or non-mutatedH3.3 peptide by IFN-γ ELISA. In (2A), among the CD8+tetramer+ population(64.1% of total lymphocyte-gated cells), there is a tetramer^(high)subpopulation (2.4% of total lymphocyte-gated cells), some of which wereused as CTL clones. In (2B), the Cap1-6D peptide (tested at 5 μg/mlonly) is a high avidity HLA-A*0201-binding epitope derived from CEA4used as an irrelevant negative control. (1C) The CTL line was evaluatedfor cytotoxicity against glioma cell lines T98 (HLA-A*0201+ butK27M-negative), HSJD-DIPG-07 (HLA-A*0201-negative but K27M+), andHSJD-DIPG-13 (HLA-A*0201+ and K27M+) lines. CFSE-labeled target cells(10e4/well) were incubated with CTLs at the E/T ratio of 25 for 4 hours.To block the CTL cytotoxicity, anti-HLA-ABC 10 μg/ml was added to onegroup. At the end of incubation, 7-ADD was added into each well andincubated for 10 minutes on ice. The samples were analyzed by flowcytometry, and the killed target cells were identified as CFSE+ and7-ADD+ cells. The cytotoxicity was calculated as the percentage of CFSE+and 7-ADD+ cells in total HLA-A*0201+ CFSE+ cells. (*p<0.05 by Wilcoxonrank-sum tests).

FIG. 3. CD8+ T cell lines stimulated with the H3.3.K27M (26-35) peptiderecognize the H3.3.K27M (26-35) but not the non-mutant counterpart. CD8+T cell lines were induced and expanded with the H3.3.K27M (26-35)peptide from the PBMCs derived from two HLA-A*0201+ healthy donors (#547and #549). T2 cells pulsed with the mutant H3.3.K27M (26-35) peptide orthe non-mutant H3.3.non-M (26-35) peptide at the indicatedconcentrations were mixed with the CD8+ T cell lines for 24 hours. IFN-γin the supernatants were assessed by ELISA.

FIG. 4. HLA-A2+ donor-derived CTLs specifically recognize HLA-A2+ K27M+glioma cells and secrete IFN-γ in an HLA-A2- and CD8-dependent manners.Peripheral blood mononuclear cells from an HLA-A2 donor were stimulatedin vitro with synthetic peptides for H3.3.K27M (26-35) and evaluatedtheir reactivity against HSID-007 (HLA-A2-negative but K27M+) orHSID-013 (HLA-A2+ and K27M+) lines by IFN-γ ELISPOT assays. Anti-HLA-ABC(10 μg/ml) and anti-CD8 antibodies were also used to evaluate whetherthe response is dependent on HLA-A2 and CD8+, respectively.

FIG. 5A-B. Characterization of H3.3.K27M-specific CTL clones. (5A)Clones were generated by limiting dilution cloning ofHLA-A2-H3.3.K27M-tetramer-positive single cells from H3.3 K27M-specificCTLs using FACS-sorting. Clones with relatively high (1C7, IH5 and 3E5)and moderate (106) affinity based on the mean fluorescence index (MFI)were selected for further evaluations. (5B) An H3.3.K27M-specific CTLclone (IH5) demonstrates H3.3.K27M-specific reactivity as shown by IFN-γELISA against T2 cells pulsed with the mutant H3.3 K27M+ peptide attitrating concentrations and the wild-type peptide (H3.3 K27M-negative;used at 500 ng/ml).

FIG. 6A-C. Evaluation of H3.3.K27M-specific TCR. (6A), J.RT-T3.5 cellswere transduced with lentiviral vector encoding the TCR α- or β-chainsderived from H3.3.K27M-specific CTL clone IH5 (J.RT-T3.5-TCR). TheJ.RT-T3.5-TCR or control non-transduced J.RT-T3.5 cells were evaluatedfor the surface TCR expression using PE-labeled HLA-A*0201/H3.3.K27Mtetramer (upper panel) or PE-labeled anti-CD3 mAb (lower panel) andFITC-labeled anti-human CD8 mAb (upper and lower panels). SinceJ.RT3-T3.5 cells are CD4+ and CD8-negative, tetramer+ CD8-negative cellsare ones expressing the transgene-derived TCR. CD3-upregulationindicates activation of cells. (6B), J.RT-T3.5-TCR, but not controlJ.RT-T3.5 cells, upregulate CD69 expression upon recognition of the H3.3K27M peptide loaded on T2 cells. (3C), DIPG 13 cells [HLA-A*0201+(albeit dim), K27M mutation+] were incubated with J.RT-T3.5-TCR orcontrol J.RT-T3.5 cells. IL-2 secretion in the culture media was assayedby specific ELISA.

FIG. 7. Expression of transgene-derived TCR. J.RT3-T3.5 cells, which aredeficient for endogenous TCR β-chain, were transduced with lentiviralvectors (pHIV-mH3TCR-IRES-Luc or pMP270-mH3TCR) encoding TCR α- andβ-chains derived from an H3.3.K27M-specific CTL clone (IH5; FIG. 5) andevaluated for the surface TCR expression using PE-labeledHLA-A2/H3.3.K27M tetramer and FITC-labeled anti-human CD8 mAb. SinceJ.RT3-T3.5 cells are CD4+ and CD8-negative, tetramer+CD8-negative cellsare ones expressing the transgene-derived TCR. Negative control cellsare non-transfected cells stained with the same tetramer indicting thespecificity of the tetramer-binding.

FIG. 8A-B. Alanine scanning to determine the key immunogenic AA residuesof the H3.3.K27M epitope. (8A) Relative HLA-A2-binding affinity of eachpeptide to that of H3.3.K27M (26-35) was determined by cell-free bindingassay using HLA-A2 purified by affinity chromatography from the EBVtransformed homozygous cell line JY. (8B), J.RT3-T3.5 cells weretransduced with lentiviral vector encoding the H3.3.K27M-specific TCRand evaluated for the recognition of each peptide loaded on T2 cells byproduction of IL-2. Each group was assayed as triplicate*<0.05 byStudent-t compared with the mutant H.3.3. In addition to 10 syntheticpeptides each containing the substitution with alanine (A1-A10), we alsoevaluated synthetic peptides designed for citrullinated H3.3. K27Mepitope (Cit H3.3; i.e., the first AA of the H3.3.K27M epitope isreplaced by citrulline) and H3.1 (a homologue of H3.3.) derived K27Mepitope (Mut H.3.1).

FIG. 9A-C. The H3.3K27M peptide is detectable by LC-MS/MS in theHLA-class I immunopeptidome of glioma cells bearing the H3.3K27Mmutation. HLA-class I peptides were biochemically purified fromU87H3.3K27M glioma cells and analyzed by LC-MS/MS with a synthetic heavyversion of the H3.3K27M peptide as the reference. 9A. U87H3.3K27MHLA-class I immunopeptidome (SEQ ID NO:2) shows two co-eluting isotopepatterns corresponding to the target m/z and mass difference of theoxidized forms of the heavy and the endogenous H3.3K27M peptides. 9B.Fragmentation spectrum of the heavy peak, showing identification of theoxidized heavy H3.3K27M peptide. 9C. Zoom-in of the light isotopepattern shows m/z values and distances between peaks as expected fromthe endogenous H3.3K27M peptide.

FIG. 10A-D. Cloning of cDNA for the H3.3K27M-specific TCR andconstruction of a retroviral vector for efficient transduction of humanT-cells. 10A. Schema of the TCR retroviral vector design. SynthesizedTCR cDNA fragments derived from the CD8+ T-cell clone 1H5 were insertedinto the Not I/Xho I site of Takara siTCR vector plasmid together withthe Kozak sequence, spacer sequence (SP) and P2A sequence. 10B. T2 cellsloaded with or without H3.3K27M peptide (10 μg/ml) were co-cultured witheither control or TCR-transduced J76CD8⁺ cells in 1:1 ratio and assessedfor IL-2 production by ELISA. Data represent three independentexperiments with similar results. *p<0.05 compared with each of othergroups. 10C. Human PBMCs were transduced with the retroviral TCR vectorand CD3⁺ T-cells were evaluated for transduction efficiency in CD8⁺ andCD8⁻ T-cell populations by the specific tetramer. 10D. TCR-transduced orcontrol CD8⁺ T-cells were co-cultured with T2 cells loaded withH3.3K27M, H3.3WT, or an irrelevant influenza matrix M1₅₈₋₆₆ peptide in1:1 ratio for 8 hrs, and evaluated for CD69 expression as an activationmarker. Dot plots represent % CD69⁺ cells among CD8⁺ T-cells. n=3 ineach group. Data represent two independent experiments with similarresults. *p<0.05 compared with each of other groups.

FIG. 11. Evaluation of TCR avidity to the HLA-A2-peptide complex. T2cells loaded with titrating concentrations of the H3.3K27M peptide(5×10³/well) were co-cultured with TCR-transduced CD8⁺ T-cells derivedfrom 3 donors (5×10³/well), and then assessed for IFN-γ secretion byELISPOT. The half-maximal effective concentration (EC₅₀) of the peptidewas calculated using non-linear regression analysis. Each experiment wascarried out in triplicate, and data represent two independentexperiments with similar results.

FIG. 12A-C. TCR-transduced T-cells lyse H3.3K27M⁺HLA-A2⁺ glioma cells inan HLA-A*0201- and H3.3K27M-dependent manner 12A and 12B. Cytotoxicityof TCR-transduced T-cells was evaluated by lactate dehydrogenase (LDH)cytotoxicity assay. Exogenous, synthetic H3.3K27M peptide (10 μg/ml) wasadded as a positive control group for TCR reactivity for each cell line.HLA-A2 blocking antibody was added in one group for each cell line todetermine the HLA-A2-dependent TCR reactivity. 12A. TCR-transduced ormock-transduced T-cells were co-cultured with H3.3K27M⁺HLA-A*0201⁺HSJD-DIPG-017 cells or control H3.3K27M⁺HLA-A*0201⁻ HSJD-DIPG-019 cellsat E/T ratio of 1, 5, and 10 for 24 hrs. 12B. TCR-transduced or controlT-cells were co-cultured with HLA-A*0201⁺ U87H3.3K27M cells or U87H3.3WTcells at E/T ratio of 5. 12C. CFSE-labeled target cells (U87H3.3K27M andU87H3.3WT cells) were co-cultured with TCR-transduced or control T-cellswith or without exogenous peptide at E/T ratio of 5. After 24 hrincubation, cells were stained with 7AAD. % CFSE⁺7AAD⁺ cells indicatedspecific percent cytotoxicity. Each group was assessed in triplicate.Data represent two independent experiments with similar results.*p<0.05, **p<0.01 based on Student's t test comparing TCR-transducedT-cells with the mock-transduced T-cells.

FIG. 13A-C. Adoptive transfer of TCR-transduced T-cells but notmock-transduced T-cells results in inhibition of intracranial H3.3K27M⁺glioma in NSG mice. NSG mice bearing intracranial U87H3.3K27Mluciferase⁺ gliomas received intravenous infusion with PBS,mock-transduced T-cells or TCR-transduced T-cells. 13A. Tumor growth ispresented as radiance (10⁷ p/s/cm²/r) using BLI (n=8 per group). Arrowsindicate days on which mice received treatment. 13B. Representative BLIimages of mice on Day 10 and on Day 32 post tumor inoculation. Thebackground BLI signals were defined based on the levels seen innon-tumor bearing mice. 13C. Preferential accumulation of TCR⁺ T-cellsin the tumor site. At the time of intravenous infusion, approximately50% and 30% of the infused CD8⁺ and CD4⁺ T-cells, respectively, wereTCR-Dextramer⁺. On Day 2 following second intravenous infusion, thepercentage of Dextramer⁺ cells among CD8⁺ T-cells and CD4⁺ T-cells wereevaluated in the peripheral blood and the brain of mice that receivedTCR-transduced T-cells. Data indicate % Dextramer⁺ cells among totallive CD8⁺ or CD4⁺ T-cells (n=5 per group). *p<0.05, **p<0.01 usingStudent t test.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The inventors have found that a peptide that encompasses amino-acidpositions 26-35 of H3.3, which includes the K27M mutation [referred toherein as “H3.3.K27M (26-35)”], can induce specific cytotoxic Tlymphocyte (CTL) responses in human leukocyte antigen (HLA)-A2+ donors.Furthermore, CTLs against H3.3.K27M (26-35) recognize HLA-A2+ gliomacell lines that also harbor the K27M mutation. Accordingly, providedherein are compositions for use in generating an immune response inhuman subjects to a peptide comprising amino-acid positions of 26-35 ofH3.3 or variants thereof.

CTL Peptides

The CTL peptides described herein comprise (R/A)MSAP(S/A)TGGV (SEQ IDNO:1), where the amino acid options in parentheses are alternativeoptions for the specified position. Accordingly, CTL peptides cancomprise RMSAPSTGGV (SEQ ID NO:2), AMSAPSTGGV (SEQ ID NO:5), RMSAPATGGV(SEQ ID NO:6), or AMSAPATGGV (SEQ ID NO:7). CTL peptides comprisingRMSAPSTGGV (SEQ ID NO:2) or AMSAPSTGGV (SEQ ID NO:5) represent peptidesfor targeting H3.3.K27M (26-35). CTL peptides comprising RMSAPATGGV (SEQID NO:6), or AMSAPATGGV (SEQ ID NO:7) represent peptides for targetingthe corresponding H3.1 K27M (26-35). The length of the CTL peptides canvary so long as the peptides are effective at inducing an immuneresponse, e.g., a CTL response. In some embodiments, the peptide willconsist of 100 or fewer or 50 or fewer amino acids. In some embodiments,the CTL peptides will have 10-14 amino acids, i.e., will be 10, 11, 12,13, or 14 amino acids long. As SEQ ID NO:1 is 10 amino acids long, the11 or 14-amino acid options will have one to four additional amino acidson the amino or carboxyl terminus of SEQ ID NO:1, or in someembodiments, one or two additional amino acid on each of the amino andcarboxyl terminus of SEQ ID NO:1. Additional amino acids can be selectedfrom any of the twenty naturally-occurring amino acids or can be anon-naturally-occurring amino acid.

The peptides can be modified to alter, for example, their in vivostability. For instance, inclusion of one or more D-amino acids in thepeptide typically increases stability, particularly if the D-amino acidresidues are substituted at one or both termini of the peptide sequence.Stability can be assayed in a variety of ways such as by measuring thehalf-life of the proteins during incubation with peptidases or humanplasma or serum. A number of such protein stability assays have beendescribed (see, e.g., Verhoef et al., Eur. J. Drug Metab. Pharmacokin.11:291-302 (1986)).

The peptides can also be modified by linkage to other molecules. Forexample, different N- or C-terminal groups may be introduced to alterthe molecule's physical and/or chemical properties. Such alterations maybe utilized to affect, for example, adhesion, stability,bioavailability, localization or detection of the molecules. Fordiagnostic purposes, a wide variety of labels may be linked to theterminus, which may provide, directly or indirectly, a detectablesignal. Thus, the peptides of the subject invention may be modified in avariety of ways for a variety of end purposes while still retainingbiological activity.

The following examples of chemical derivatives are provided by way ofillustration and not by way of limitation.

Aromatic amino acids may be replaced with D- or L-naphylalanine, D- orL-Phenylglycine, D- or L-2-thieneyl-alanine, D- or L-1-, 2-, 3- or4-pyreneylalanine, D- or L-3-thienylalanine, D- or L-(2-pyridinyl)-alanine, D- or L- (3-pyridinyl)-alanine, D- or L-(2-pyrazinyl)-alanine, D- or L- (4-isopropyl)-phenylglycine,D-(trifluoromethyl)-phenyl-glycine, D-(trifluoromethyl)-phenylalanine,D-p-fluoro-phenylalanine, D- or L-p-biphenylphenylalanine, D- orL-p-methoxybiphenylphenylalanine, D- or L-2-indole-(alkyl)alanines, andD- or L-alkylamines where alkyl may be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, iso-propyl, iso-butyl,sec-isotyl, iso-pentyl, non-acidic amino acids, of C1-C20.

Acidic amino acids can be substituted with non-carboxylate amino acidswhile maintaining a negative charge, and derivatives or analogs thereof,such as the non-limiting examples of (phosphono)-alanine, glycine,leucine, isoleucine, threonine, or serine; or sulfated (e.g., —SO₃H)threonine, serine, tyrosine.

Other substitutions may include unnatural hydroxylated amino acids madeby combining “alkyl” (as defined and exemplified herein) with anynatural amino acid. Basic amino acids may be substituted with alkylgroups at any position of the naturally occurring amino acids lysine,arginine, ornithine, citrulline, or (guanidino)-acetic acid, or other(guanidino) alkyl-acetic acids, where “alkyl” is define as above.Nitrile derivatives (e.g., containing the CN-moiety in place of COOH)may also be substituted for asparagine or glutamine, and methioninesulfoxide may be substituted for methionine. Methods of preparation ofsuch peptide derivatives are well known to one skilled in the art.

In addition, any amide linkage can be replaced by a ketomethylenemoiety, e.g., (—C(═O)—CH₂—) for (—(C═O)—NH—). Such derivatives areexpected to have the property of increased stability to degradation byenzymes, and therefore possess advantages for the formulation ofcompounds which may have increased in vivo half-lives, as administeredby oral, intravenous, intramuscular, intraperitoneal, topical, rectal,intraocular, or other routes.

In addition, any amino acid can be replaced by the same amino acid butof the opposite chirality. Thus, any amino acid naturally occurring inthe L-configuration (which may also be referred to as the R or Sconfiguration, depending upon the structure of the chemical entity) maybe replaced with an amino acid of the same chemical structural type, butof the opposite chirality, generally referred to as the D-amino acid butwhich can additionally be referred to as the R— or the S—, dependingupon its composition and chemical configuration. Such derivatives havethe property of greatly increased stability to degradation by enzymes,and therefore are advantageous in the formulation of compounds which mayhave longer in vivo half-lives, when administered by oral, intravenous,intramuscular, intraperitoneal, topical, rectal, intraocular, or otherroutes.

Fusion peptides including an antigenic peptide as described above fusedto another peptide sequence are specifically contemplated for use withthe methods and compositions described herein. In some embodiments,peptides include fusion peptides composed of a CTL sequence as describedherein (e.g., SEQ ID NO:1) and a helper T lymphocyte (CD4) epitopesequence fused together. Examples of suitable CD4 epitopes include thesynthetic sequence PADRE, tetanus-specific peptides, peptides derivedfrom the same antigen or other antigens from the virus that is to betargeted. Similarly, for cancer antigens, a CD4 peptide derived from thesame antigen, or any other cell-antigen known in the art, and the likemay be used. Linker peptide sequences at the N- or C-terminal end of thefusion or between the CTL and CD4 epitopes in the fusion also may beused. Such linker sequences generally can be, for example, from about 1to about 10 amino acids in length and, e.g., about 2 to about 7 aminoacids or from about 3 to about 5 amino acids in length can optionallycomprise modified or non-traditional amino acids.

Also provided are peptide/HLA-A2 multimers, and in some embodiments,their use to isolate peptide-specific CTLs. “Multimers” include, e.g.,tetramers, pentamers and any number of MHC-peptide structure assembledaround the core molecule with fluorochrome. In some embodiments, theselected MHC molecules (e.g., HLA-A2) along with beta2-microgloblin areassembled around one core molecule which connects a fluorochromemolecule (such as FITC, so that the multimer will fluoresce) andmultiple MHC-beta-microgloblin molecules (in case of tetramer andpentamer, there will be 4 and 5, respectively). Then, the MHC moleculeis also bound with the peptide so that the MHC-peptide complex on themultimer molecule can be recognized by the TCR on T-cells. See, e.g.,Wooldridge, et al., Immunology 126(2): 147-164 (2009); Yokouchi, et al.,Cancer Sci. 97(2):148-54 (2006). Accordingly, in some embodiments,fluorochrome-conjugated peptide-major histocompatibility complex (pMHC)multimers, wherein the peptide is a CTL peptide (e.g., comprising SEQ IDNO:1) are provided.

In some embodiments, T-cell populations are enriched for T-cellsexpressing one or more TCRs that bind to the CTL peptide/MHC complex.For example, in some embodiments, a T-cell population is cultured in thepresence of the CTL peptide (either the naked peptide or peptide loadedonto APCs), thereby preferentially stimulating division of T-cellscarrying TCRs that bind the peptide/MHC complex. In some embodiments,the culturing occurs in the presence of IL-2, IL-4, IL-7 or IL-15 alone,or of 2-way, 3-way, or 4-way combinations thereof. T-cell populationsexpanded in this way will be enriched for TCRs that bind the protein/MHCcomplex. Subsequently, fluorochrome-conjugated peptide-majorhistocompatibility complex (pMHC) multimers can be used to label theT-cells expressing the TCRs that bind the peptide/MHC complex, and canbe sorted, for example by FACS.

Exemplary TCR alpha and beta chain sequences that recognize theH3.3.K27M epitope are provided below with CDR1, CDR2, and CDR3underlined.

TCRA-Val9*01/J43 (SEQ ID NO: 8)Met L T A S L L R A V I A S I C V V S S Met A Q K V T Q A Q T E I S V V E K E D V TL D C V Y E 

 Y L F W Y K Q P P S G E L V F L I R

 D E Q N E I S GR Y S W N F Q K S T S S F N F T I T A S Q V V D S A V Y F C 

 E N D Met R F GA G T R L T V K P N I Q N P D P A V Y Q L R D S K S S D K S V C L F T D F D S Q T NV S Q S K D S D V Y I T D K T V L D Met R S Met D F K S N S A V A W S N K S D F AC A N A F N N S I I P E D T F F P S P E S S C D V K L V E K S F E T D T N L N F Q N LS V I G F R I L L L K V A G F N L L Met T L R L W S S Stop CDR1:(SEQ ID NO: 12) T R D T T Y Y; CDR2: (SEQ ID NO: 13) R N S F CDR3:(SEQ ID NO: 14) A L S ECoding sequence of the above amino acid sequence: (SEQ ID NO: 9)ATGCTGACTGCCAGCCTGTTGAGGGCAGTCATAGCCTCCATCTGTGTTGTATCCAGCATGGCTCAGAAGGTAACTCAAGCGCAGACTGAAATTTCTGTGGTGGAGAAGGAGGATGTGACCTTGGACTGTGTGTATGAAACCCGTGATACTACTTATTACTTATTCTGGTACAAGCAACCACCAAGTGGAGAATTGGTTTTCCTTATTCGTCGGAACTCTTTTGATGAGCAAAATGAAATAAGTGGTCGGTATTCTTGGAACTTCCAGAAATCCACCAGTTCCTTCAACTTCACCATCACAGCCTCACAAGTCGTGGACTCAGCAGTATACTTCTGTGCTCTGAGTGAGGAGAATGACATGCGCTTTGGAGCAGGGACCAGACTGACAGTAAAACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA TCRB-Vb27/J2.7(SEQ ID NO: 10)Met G P Q L L G Y V V L C L L G A G P L E A Q V T Q N P R Y L I T V T G K K L T VT C S Q 

 Met S W Y R Q D P G L G L R Q I Y 

 D K G D V P E G Y K V S R K E K R N F P L I L E S P N P N Q T S L Y F 

 F G P G T R L T V T E D L K N V F P P E V A V F E P S E A E I S H T Q K A T LV C L A T G F Y P D H V E L S W W V N G K E V H S G V S T D P Q P L K E Q P A LN D S R Y C L S S R L R V S A T F W Q N P R N H F R C Q V Q F Y G L S E N D E W TQ D R A K P V T Q I V S A E A W G R A D C G F T S E S Y Q Q G V L S A T I L Y E I LL G K A T L Y A V L V S A L V L Met A Met V K R K D S R G Stop CDR1:(SEQ ID NO: 15) N Met N H E Y; CDR2: (SEQ ID NO: 16) Y S Met N V E V TCDR3: (SEQ ID NO: 17) C A S G W G G P F Y E Q YCoding sequence of the above amino acid sequence: (SEQ ID NO: 11)ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATCCTGGAGTCGCCCAACCCCAACCAGACCTCTCTGTACTTCTGTGCCAGCGGCTGGGGTGGTCCATTCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG

In some embodiments, one or more T-cells expressing TCR alpha and betachain sequences that recognize the H3.3.K27M epitope (e.g., as presentedin an antigen presenting cell in the context of an MHC/HLA protein) areadministered to an individual (e.g., a human) In some embodiments, theindividual has one or more cells that express the H3.3.K27M epitope andthe T-cell is administered to contact the cell expressing the epitope.In some embodiments, the T-cell, once in contact with the cellexpressing the epitope, directly or indirectly kills the cell. In someembodiments, the cell expressing the epitope is a glioma cell. In someembodiments, the glioma cell is a glioblastoma (GBM) cell. In someembodiments, the glioma cell is a diffuse intrinsic pontine glioma(DIPG) cell. In some embodiments, the glioma cell is an ependymoma,astrocytoma, oligodendroglioma, brainstem glioma, thalamic glioma,spinal cord glioma, or optic nerve glioma. In some embodiments, theT-cells are administered intracranially or intravenously.

In some embodiments, the T-cell is a primary or expanded T-cell. In someembodiments, the T-cells are CD4⁺ T-cells or CD8 T-cells. In someembodiments, the T-cell is from the individual into which an expressioncassette encoding the TCR or part thereof has been introduced so thatthe T-cell expresses the TCR on the surface of the T-cell. Methods ofgenerating T-cells expressing heterologous genes and methods ofadministering T-cells are described in, for example, US PatentPublications 2017/0067021 and 2016/0120905.

Polynucleotides

Polynucleotides encoding the CTL peptides described herein are providedand are referred to as “CTL polynucleotides”. In some embodiments, theCTL polynucleotides can be a DNA or RNA sequence. The CTL polynucleotideis can be operably linked to some or all of transcriptional andtranslational regulatory elements, such as a promoter, enhancer andpolyadenylation sequence. Regulatory sequences are art-recognized andare described, e.g., in Goeddel; Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif.; (1990). In someembodiments, the promoter is a constitutive promoter, e.g., a strongviral promoter, e.g., CMV promoter. The promoter can also be cell- ortissue-specific, that permits substantial transcription of the DNA onlyin predetermined cells, e.g., in antigen presenting cells, such as thedendritic cell-specific CD11c promoter described in Brocker T, J. Leuk.Biology 66:331-335, 1999. The promoter can also be an induciblepromoter, for example, a metallothionein promoter. Other induciblepromoters include those that are controlled by the inducible binding, oractivation, of a transcription factor, e.g., as described in U.S. Pat.Nos. 5,869,337 and 5,830,462 by Crabtree et al., describing smallmolecule inducible gene expression (a genetic switch); Internationalpatent applications PCT/US94/01617, PCT/US95/10591, PCT/US96/09948 andthe like, as well as in other heterologous transcription systems such asthose involving tetracyclin-based regulation reported by Bujard et al.,generally referred to as an allosteric “off-switch” described by Gossenand Bujard, Proc. Natl. Acad. Sci. U.S.A. (1992) 89:5547 and in U.S.Pat. Nos. 5,464,758; 5,650,298; and 5,589,362 by Bujard et al. Otherinducible transcription systems involve steroid or other hormone-basedregulation.

The CTL polynucleotides may also be produced in part or in total bychemical synthesis, e.g., by the phosphoramidite method described byBeaucage and Carruthers, Tetra. Letts., 22:1859-1862 (1981) or thetriester method according to the method described by Matteucci et al.,J. Am. Chem. Soc, 103:3185 (1981), and may be performed on commercialautomated oligonucleotide synthesizers. A double-stranded fragment maybe obtained from the single-stranded product of chemical synthesiseither by synthesizing the complementary strand and annealing the strandtogether under appropriate conditions or by adding the complementarystrand using DNA polymerase with an appropriate primer sequence.

The CTL polynucleotide operably linked to all necessary transcriptionaland translational regulation elements can be injected as naked DNA intoa subject or contacted in vitro with antigen presenting cells. In someembodiments, the CTL polynucleotide and regulatory elements are presentin a plasmid or vector. Thus, the CTL polynucleotide can be DNA, whichis itself non-replicating, but is inserted into a plasmid, which mayfurther comprise a replicator. The DNA can be a sequence engineered soas not to integrate into the host cell genome. Exemplary vectors areexpression vectors, i.e., vectors that allow expression of a nucleicacid in a cell. Exemplary expression vectors are those which containboth prokaryotic sequences, to facilitate the propagation of the vectorin bacteria, and one or more eukaryotic transcription units that areexpressed in eukaryotic cells.

Alternatively, derivatives of viruses such as the bovine papillomaviras(BPV-1), of Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Theseviruses expressing the CTL polypeptides can be used to infect APCs,which are then administered to patients. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning: A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989), Chapters 16 and 17.

In some embodiments, the CTL polynucleotide is expressed in aprokaryote. In some embodiments, the polypeptide is expressed in E.coli. In some embodiments, the CTL polypeptide is expressed in Listeriamonocytogenes, which in some embodiments, is attenuated, and which canbe administered to a human individual to provide the CTL peptide to theindividual. See, e.g., US Patent Publication No. US2013/0259891. Inother embodiments, the CTL polynucleotide is expressed in a eukaryoticcell. For example, the CTL polynucleotide and polypeptide can beexpressed in yeast, insect cells, animal cells, including mammaliancells, e.g., human cells.

Inducing an Immune Response

The CTL peptides of the invention are capable of inducing an immuneresponse when administered to an animal (e.g., a human). An exemplaryimmune response is a CTL response. The CTL peptides, once introduced canbe processed and presented to the MHC class I complex of an antigenpresenting cell. Human MHC class I complex includes HLA-A, B, and Calleles. The CTL peptides (e.g., SEQ ID NO:1) have higher affinity toHLA-A2+ individuals. See Table 1. The MHC class I-bound epitope is thentransported to the cell surface and recognized by cytotoxic Tlymphocytes (CTLs) through T cell receptors (TCRs) located on theirsurface. Recognition of an antigen/MHC complex by the TCR triggers acascade of protein and cytokine interactions leading to, among otherinteractions, the activation, maturation and proliferation of theprecursor CTLs, resulting in CTL clones capable of destroying the cellsexhibiting CTL peptides recognized as foreign.

In one embodiment, one or more CTL peptides as described herein areadministered to the patient. In another embodiment, one or morepolynucleotides encoding one or more CTLs are introduced to APCs, andthese APCs expressing CTLs are administered into a human patient toinduce an immune response, the cytotoxic T cell response. In someembodiments, the CTL peptides are loaded onto the APCs withoutexpressing the CTL peptides in the cell. See, e.g., U.S. Pat. No.8,652,462. In some embodiments, the APCs are harvested from anindividual, loaded with the CTL peptides (or the CTL polynucleotides areintroduced into the APCs), and then the APCs are introduced back intothe individual (i.e., the cells are autologous) and a CTL immuneresponse is induced in the individual to the CTL polypeptide.

In one embodiment, an immune response is induced by directlyadministering the CTL peptides to patients. Adjuvants and/or nonspecificinflammatory mediators are not required, but can be optionallyadministered with the active ingredient before, during, or after thepriming and/or boosting of the immune response. Adjuvants are substancesthat are used to specifically or nonspecifically potentiate anantigen-specific immune response, perhaps through activation of antigenpresenting cells. The adjuvant can be, e.g., Montanide-ISA 51 (e.g.,from Seppic Hiltonol (e.g., from Oncovir), anti-CD40 agonisticmonoclonal antibodies, polyICLC, Bacillus Calmette-Guérin (BCG) vaccine,an ADP-ribosylating exotoxin (e.g., cholera toxin, diphtheria toxin, E.coli heat-labile enterotoxin, pertussis toxin, P. aeruginosa exotoxinA), a fragment thereof containing the A and/or B subunit, a chemicallymodified or genetically mutated derivative thereof, or a derivativethereof with reduced toxicity; a chemical conjugate or geneticrecombinant containing a bacterial ADP-ribosylating exotoxin orderivative thereof; a chemokine (e.g., defensins, HCC-1, HCC-4, MCP-1MCP-3, MCP-4, MLP-1α, MIP-1β, MIP-1γ, MIP-3α, MIP-2, RANTES); anotherligand of a chemokine receptor (e.g., CCR1, CCR-2, CCR-5, CCR-6,CXCR-1); a cytokine (e.g., IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12; IFN-γ;TNF-α; GM-CSF); another ligand of a cytokine receptor; a salt (e.g.,aluminum hydroxide or phosphate, calcium phosphate); lipid A or aderivative thereof (e.g., monophosphoryl or diphosphoryl lipid A, lipidA analogs, AGP, ASO2, ASO4, DC-Choi, Detox, OM-174); apathogen-associated molecular pattern (PAMP); immunostimulatory CpGmotifs in bacterial DNA or an oligonucleotide (see, for example, U.S.Pat. No. 6,218,371); a Leishmania homolog of elF4a or a derivativethereof (see, for example, U.S. Pat. No. 5,876,735); a heat shockprotein or derivative thereof; C3d tandem array; a muramyl dipeptide(MDP) or a derivative thereof (e.g., murabutide, threonyl-MDP, muramyltripeptide); ISCOMS and saponins (e.g., Quil A, QS-21); squalene;superantigens; a ligand of a toll-like receptor, or the like. Adjuvantsmay be chosen to preferentially induce antibody or cellular effectors,specific antibody isotypes (e.g., IgM, IgD, IgA1, IgA2, secretory IgA,IgE, IgG1, IgG2, IgG3, and/or IgG4), or specific T-cell subsets (e.g.,CTL, Th1, Th2 and/or TDTH)—For example, antigen presenting cells maypresent Class Il-restricted antigen to precursor CD4+ T cells, and theTh1 or Th2 pathway may be entered.

In another embodiment, the invention provides a method of administeringAPC cells expressing and/or presenting CTL peptides to induce immuneresponse. Methods for delivering CTL polynucleotide to APCs arewell-known in the art, for example, retroviruses, adenoviruses,lentiviruses, adeno-associated virus (AAV), and herpes simplex virus-1,vaccinia viruses, and canarypox or fowlpox viruses can infect APC. Mostof these vectors cause transient gene expression lasting less than twoweeks (Bonnet et al, 2000). To initiate an immune response, expressionof peptides, proteins, or MHC molecules may only need to last aboutthree to ten days (see, e.g., U.S. Pat. Nos. 5,656,465 and 5,833,975).Plasmids may also be used to transfer a gene into the cell by itself orwith chemicals that enhance transfection, or via carriers. For example,cationic lipids, calcium phosphate, DEAE-dextran, polybrene-DMSO, orpolycation amino acids (e.g., polylysine) are chemical transfectants.

The method may further involve a targeting molecule that preferentiallybinds to a target cell, for example antigen presenting cells. Suchtargeting mechanisms may include terminal galactosyl residues binding toasialoglycoprotein receptor (e.g., galactosylated nucleic acid and/orantigen), high-mannose oligosaccharide binding to mannose receptor(e.g., mannosylated nucleic acid and/or antigen), ligand binding to anFc receptor (e.g., nucleic acid and/or antigen fused or linked to IgGconstant region or other ligand of CD64), or membrane proteins highlyexpressed on antigen presenting cells (e.g., nucleic acid and/or antigenfused or linked to ligand or antibody specific for the membraneprotein). Mannose receptors are exemplary targets because they arehighly expressed on dendritic cells (especially Langerhans cells), andinvolved in antigen uptake. This significantly increases the APC'sability to capture exogenous proteins and process them (Sallusto, 1995).Antigen may be delivered to phagocytic cells of the skin such as, forexample, Langerhans cells, other dendritic cells, macrophages, and otherantigen presenting cells in the epidermis and dermis; antigen may alsobe delivered to phagocytic cells of the liver, spleen, and bone marrowthat are known to serve as the antigen presenting cells through theblood stream or lymphatic system.

In a further embodiment, the composition can comprise the CTLpolypeptide and also comprise the universal T cell epitope called PADRE®(Epimmune, San Diego; described, for example in U.S. Pat. No. 5,736,142or International Application WO95/07707, which are enclosed herein byreference). A “PanDR binding peptide” or “PADRE® peptide” is a member ofa family of molecules that binds more than one HLA class II DR molecule.The pattern that defines the PADRE® family of molecules can be thoughtof as an HLA Class U supermotif. PADRE® binds to most HLA-DR moleculesand stimulates in vitro and in vivo human helper T lymphocyte (HTL)responses. Alternatively, T-helper epitopes can be used from universallyused vaccines such as tetanus toxoid.

Typically, a vaccine or vaccine composition is prepared as aninjectable, either as a liquid solution or suspension. Injection may besubcutaneous, intramuscular, intravenous, intraperitoneal, intrathecal,intradermal, intraepidermal, or by “gene gun”. Other types ofadministration comprise electroporation, implantation, suppositories,oral ingestion, enteric application, inhalation, aerosolization or nasalspray or drops. Solid forms, suitable for dissolving in or suspensionin, liquid vehicles prior to injection may also be prepared. Thepreparation may also be emulsified or encapsulated in liposomes forenhancing adjuvant effect.

A liquid formulation may include oils, polymers, vitamins,carbohydrates, amino acids, salts, buffers, albumin, surfactants, orbulking agents. Exemplary carbohydrates include sugar or sugar alcoholssuch as mono-, di-, or polysaccharides, or water-soluble glucans. Thesaccharides or glucans can include fructose, dextrose, lactose, glucose,mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin,alpha- and beta-cyclodextrin, soluble starch, hydroxyethyl starch andcarboxymethylcellulose, or mixtures thereof. “Sugar alcohol” is definedas a C4 to C8 hydrocarbon having an —OH group and includes galactitol,inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. Thesesugars or sugar alcohols mentioned above may be used individually or incombination. There is no fixed limit to the amount used as long as thesugar or sugar alcohol is soluble in the aqueous preparation. In someembodiments, the sugar or sugar alcohol concentration is between 1.0%(w/v) and 7.0% (w/v), e.g., between 2.0 and 6.0% (w/v). Exemplary aminoacids include levorotary (L) forms of carnitine, arginine, and betaine;however, other amino acids may be added. Exemplary polymers includepolyvinylpyrrolidone (PVP) with an average molecular weight between2,000 and 3,000, or polyethylene glycol (PEG) with an average molecularweight between 3,000 and 5,000. In some embodiments, one can use abuffer in the composition to minimize pH changes in the solution beforelyophilization or after reconstitution. Any physiological buffer may beused, but in some cases can be selected form citrate, phosphate,succinate, and glutamate buffers or mixtures thereof. Surfactants thatcan be added to the formulation are shown in EP patent applications No.EP 0 270 799 and EP 0 268 110.

Additionally, polypeptides can be chemically modified by covalentconjugation to a polymer to increase their circulating half-life, forexample. Exemplary polymers, and methods to attach them to peptides, areshown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546.Exemplary polymers are polyoxyethylated polyols and polyethylene glycol(PEG). PEG is soluble in water at room temperature and has the generalformula: R(O—CH₂—CH₂)_(n)O—R where R can be hydrogen, or a protectivegroup such as an alkyl or alkanol group. In some embodiments, theprotective group has between 1 and 8 carbons, e.g., methyl. The symbol nis a positive integer, preferably between 1 and 1000, e.g., between 2and 500. The PEG can have, for example, average molecular weight between1000 and 40,000, e.g., between 2000 and 20,000, e.g., between 3,000 and12,000. In some embodiments, PEG has at least one hydroxyl group, e.g.,it has a terminal hydroxy group.

Water soluble polyoxyethylated polyols are also useful and can be linkedto the CTL polypeptides described herein. They include polyoxyethylatedsorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG),etc. Another drug delivery system for increasing circulatory half-lifeis the liposome. The peptides and nucleic acids of the invention mayalso be administered via liposomes, which serve to target a particulartissue, such as lymphoid tissue, or to target selectively infectedcells, as well as to increase the half-life of the peptide and nucleicacids composition. Liposomes include emulsions, foams, micelles,insoluble monolayers, liquid crystals, phospholipid dispersions,lamellar layers and the like. Liposomes either filled or decorated witha desired peptide or nucleic acids of the invention can be directed tothe site of lymphoid cells, where the liposomes then deliver the peptideand nucleic acids compositions. Liposomes for use in accordance with theinvention are formed from standard vesicle-forming lipids, whichgenerally include neutral and negatively charged phospholipids and asterol, such as cholesterol. The selection of lipids is generally guidedby consideration of, e.g., liposome size, acid lability and stability ofthe liposomes in the blood stream. A variety of methods are availablefor preparing liposomes, as described in, e.g., Szoka et al., 1980, andU.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporatedinto the liposome can include, e.g., antibodies or fragments thereofspecific for cell surface determinants of the desired immune systemcells. A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc., in a dose which variesaccording to, inter alia, the manner of administration, the peptidebeing delivered, and the stage of the disease being treated.

After the liquid pharmaceutical composition is prepared, the compositioncan be lyophilized to prevent degradation and to preserve sterility.Just prior to use, the composition may be reconstituted with a sterilediluent (Ringer's solution, distilled water, or sterile saline, forexample) which may include additional ingredients. Upon reconstitution,the composition can be administered to subjects.

The polypeptide and APCs of the invention can be used to treatindividuals having gliomas. In some embodiments, the gliomas areselected from glioblastoma (GBM) and diffuse intrinsic pontine gliomas(DIPG). In some embodiments, the individual is a HLA-A2+ type. The HLAgenes are the human versions of the major histocompatibility complex(MHC) genes that are found in most vertebrates (and thus are the moststudied of the MHC genes). HLAs corresponding to MHC class I are HLA-A,HLA-B, and HLA-C. HLAs corresponding to MHC class II are DP, DM, DOA,DOB, DQ, and DR. Tests for HLA typing are readily available and can beused to screen patients who are likely benefit from the treatment of theinvention, for example, from the world wide web atlabtestsonline.org/understanding/analytes/hla-testing/tab/test/. In somecases, the patient may also have clinical symptoms besides gliomas. Theglioma patient may belong to any age group, including children (e.g.,0-18 years old).

Antibodies Recognizing the CTL Peptides

Also provided is an antibody or an antigen-binding fragment thereof,that recognizes the CTL peptides described herein. Antigen-bindingfragments of antibodies encompass fragments which comprise thehypervariable domains designated CDRs (Complementarity DeterminingRegions) or part(s) thereof encompassing the recognition site for theantigen, i.e., the CTL peptides described herein, thereby definingantigen recognition specificity. Each Light and Heavy chain(respectively VL and VH) of a four-chain immunoglobulin has three CDRs,designated VL-CDR1, VL-CDR2, VL-CDR3 and VH-CDR1, VH-CDR2, VH-CDR3,respectively. Thus, in some cases the antibody comprises fragments ofantibodies of the invention (antigen-binding fragments), which compriseor consist of all or a selection of CDRs among VL-CDR1, VL-CDR2, VL-CDR3and VH-CDR1, VH-CDR2 and VH-CDR3 or functional portions thereof, i.e.portions that exhibit the desired binding specificity, preferably with ahigh affinity, for the CTL peptides of the invention. For example, insome embodiments, the antibody or antibody fragment binds to SEQ ID NO:2but does not bind to (e.g., binds at no higher than background) SEQ IDNO:3.

The antibody may be produced by any well-known method for antibodyproduction. For example, the antibody may be obtained by administrationof any of the above embodiments of CTL peptides into an animal andharvesting the antibodies produced as a result.

EXAMPLES Example 1

The H3.3.K27M-Derived HLA-A*0201-Restricted Cytotoxic T-Lymphocyte (CTL)Epitope and Cloning of T-Cell Receptor (TCR) cDNAs

1. Screening of HLA-A*0201-Binding Epitopes in H3.3 with the K27MMutation

Using the NetMHC 3.4 server (on the world wide web atcbs.dtu.dk/services/NetMHC/), an artificial neural network-basedprediction system of peptide binding motifs to HLAs, we predicted thatan H3.3-derived 10-mer amino acid (AA) peptide which encompasses the AAposition 26-35 including the K27M mutation [1-13.3.K27M (26-35)] willserve as a potent epitope in the context of HLA-A*0201, while thenon-mutant counterpart for the corresponding positions [1-13.3.Non-M(26-35)] was not predicted to have high affinity to HLA-A*0201 (Table1).

TABLE 1 The H3.3 (26-35) peptide Binding Scores logscore affinity(nM)K27M Mutant: RMSAPSTGGV 0.477 285 Non-mutant: RKSAPSTGGV 0.073 22651

Analysis of H3.3.K27M (26-35), H3.3.Non-M (26-35) peptides as well as9-mer peptides H3.3.K27M (19-27) and H3.3.Non-M (19-27). We nextdirectly evaluated relative binding affinity of these peptides using thetransporter associated with antigen processing (TAP)-deficientHLA-A*0201+ T2 cell line. Since stable binding of HLA-A*0201 withpeptide epitopes further stabilizes the surface expression ofHLA-A*0201, quantitative expression levels of HLA-A*0201 correlate withthe binding affinity of the peptide-epitopes that are co-incubated withthe T2 cells (FIG. 1). The MFI values for T2 cells with no peptideindicate the baseline HLA-A2 expression level. Cap6D is awell-documented HLA-A*0201-binding epitope and used as a positivecontrol for the assay. The H3.3.K27M (26-35), but none of otherH3.3-derived peptides, demonstrated a peptide-dose-dependent increase ofmean fluorescence intensity (MFI). These data suggested that H3.3.K27M(26-35) peptide is a potential epitope for specific T-cell responses. Toprecisely measure the binding of peptides to HLA-A*02:01 class Imolecules, we performed a competitive binding inhibition assay, andevaluated the concentration of peptide yielding 50% inhibition of thebinding of the radiolabeled probe peptide (IC50). The mutant H3.3.K27Mpeptide constantly demonstrated IC50 of 95 nM-187 nM in 4 separatesamples, which are considered to be excellent binding capabilities. Onthe other hand, the non-mutant H3.3 peptide (on the correspondingposition) demonstrated >30 fold lower binding capabilities. These dataconfirm the high HLA A*02:01-binding capability of the H3.3.K27Mpeptide.

Then, we stimulated HLA-A*0201 donor-derived peripheral bloodmononuclear cells (PBMCs) with synthetic peptide for H3.3.K27M (26-35)in vitro for several weekly cycles and evaluated for the induction ofCD8+ T-cells capable of binding the HLA-A*0201-H3.3.K27M (26-35)tetramer, and found over 60% of CD8+ cells bound to the tetramer (FIG.2A). Furthermore, a subpopulation of the total CD8+ cells in the cultureshowed distinctively higher levels of binding to the tetramer,suggesting that these are high-affinity binders among the H3.3.K27M(26-35)-reactive T-cell populations.

2. Antigen-Specific IFN-γ Production and Lytic Activity of H3.3.K27M(26-35)-Stimulated CD8+ T-cells.

CD8+ T cell lines that had been stimulated with the H3.3.K27M (26-35)peptide demonstrate peptide-dose-dependent increases of IFN-γ productionin response to T2 target cells loaded with H3.3.K27M (26-35) compared toT2 cells loaded with the control H3.3.Non-M (26-35), cap6D peptide ornothing (FIGS. 2B and 3).

3. H3.3.K27M (26-35)-Stimulated CD8+ T-Cells Recognize the CognateEpitope Endogenously Expressed in HLA-A*0201 Glioma Cells.

We next examined whether the CD8+ T-cell line developed in response tothe H3.3.K27M (26-35) peptide recognize HLA-A*0201+ human glioma cellsthat endogenously express and present the H3.3.K27M (26-35) epitope. Weused HSID-007 (HLA-A*0201-negative but K27M+) or HSID-013 (HLA-A*0201+and K27M+) as target glioma cells. As illustrated in FIG. 2C and FIG. 4,the CD8+ T-cell line responded to HSID-013 but not to HSID-007 cellsbased on cytotoxic T-lymphocyte (CTL) assays (FIG. 2C). Furthermore, theobserved response against HSID-013 cells was almost completely blockedby anti-HLA-class I blocking antibody. These data are notable becausethey indicate that the CD8+ T-cell lines (we have similar data for twoadditional cell lines; but only one is shown here) recognize theH3.3.K27M (26-25) mutant epitope endogenously expressed and presented byHLA-A*0201+ glioma cell lines.

4. High Affinity H3.3.K27M-Specific CTL Clones.

We have obtained CTL clones that are reactive to the H3.3.K27M epitope(FIG. 5A-B). These clones retain specificity to the H3.3. K27M epitopeand we have isolated T-cell receptor (TCR) α and β chains from the clone1H5.

5. Cloning of H3.3.K27M-Specific T-Cell Receptor cDNA.

We have cloned full-length α- (SEQ ID NO:8) and β-chains (SEQ ID NO:10)of TCRs from an H3.3.K27M-specific CD8+ T-cell clone (1H5), andconstructed a lentiviral vector encoding the α- and β-chains. As thefirst step to confirm expression of the transgene-TCR, we transducedJ.RT3-T3.5 cells, which are deficient for endogenous TCR β-chain, withthe lentiviral vector and evaluated for expression ofHLA-A*0201/H3.3.K27M-tetramer-reactive TCR by flow cytometry (FIG. 6A;upper panels; FIG. 7). More than 60% of J.RT3-T3.5 cells showed positivetetramer binding, whereas only 0.69% of non-transduced J.RT3-T3.5 cellsshowed the signal, indicating that the transgene TCR is expressed byboth lentiviral constructs. In the same setting, we also observed thatTCR-transduced cells, but not control cells upregulate CD3 expressionwhen co-incubated with the tetramer (FIG. 6A lower panels). These cellsalso upregulate CD69 as an indication of activation in apeptide-dose-dependent, and TCR-specific manner (FIG. 6B). These dataclearly indicate antigen-specific reactivity of the TCR when transducedin J.RT3-T3.5 cells.

Finally, TCR-transduced cells, but not control cells, were able toproduce IL-2 when they were co-cultured with HLA-A*0201+, H3.3.K27M+DIPG-013 cells (FIG. 6C).

6. Absence of Detectable Deiminated H3 Protein in Cultured Glioma Cells

The natural process of deimination can convert histone arginine tocitrulline, including the arginine residue within the H3.3.K27M epitope.Therefore, it is useful to determine whether glioma cells undergodeimination, and, if so, whether the replacement of the arginine residuewithin the H3.3.K27M epitope with citrulline (i.e. deimination) impactsthe immunogenicity of the H3.3.K27M epitope.

We performed Western blot analyses to determine whether H3.3.K27M⁺glioma cells have deiminated H3 protein using a mAb specific todeiminated H3 (abcam cat # ab19847). While neutrophils stimulated withcalcium ionophore as the positive control sample showed a single band ofapproximately 15 kDa corresponding to the deiminated H3, none of theglioma cell lines demonstrated a similar band, strongly suggesting thatglioma cell lines are not substantially deiminated, at least whencultured in vitro. The positive control sample is from neutrophilsstimulated with calcium ionophore. Glioma cells evaluated areHSJD-DIPG-13, HSJD-DIPG-12, HSJD-DIPG-08, HSJD-DIPG-07, SU06, SU-D-04,T98, and U87. Each lane was loaded with 20 μg protein lysate and highquality SDS-PAGE was confirmed each time by Coomassie Blue staining. Werepeated these Western blot analyses at least 4 times (4 SDS-PAGE runs)with different incubation and exposure conditions, and found a thin bandwith glioma cells at very high exposure conditions (not shown),suggesting that deimination may occur in cultured glioma cells at lowlevels.

7. Alanine Scanning Data Suggests that there are No Known Human Proteinsthat Share the Key Immunogenic AA Residues of the H3.3.K27M Epitope

To ensure the specificity and safety of the H3.3.K27M-targetingapproach, it is useful to determine key AA residues in the H3.3.K27Mepitope that are responsible for the CTL reactivity. This will allowprecise predictions and assessments for cross-reactivity to otherepitopes derived from proteins in non-tumor normal cells.

To this end, alanine-scanning mutagenesis was used. Single alaninemutations (10 in total) were introduced at every AA residue within theH3.3.K27M decamer (10-mer) epitope. Hence, 10 synthetic peptides eachcontaining the specific substitution with alanine were prepared(A1-A10). Using each of the synthetic peptides withalanine-substitutions, we evaluated whether the substitution alters thestability of peptide-binding to HLA-A*0201 (FIG. 8A). When the bindingis diminished by the substitution compared with the natural H3.3.K27Mepitope (“Mut H3.3.” in FIG. 8A-B), it indicates that the substitutedresidue was critical for HLA-A*0201-binding. We also evaluated whetherthe H3.3.K27M-specific TCR recognizes the altered epitopes presented onT2 cells. To this end, we evaluated IL-2 production as the readout forthe activation of TCR-transduced J.RT3-T3.5 cells when co-cultured withT2 cells loaded with each of the altered peptides (FIG. 8B). AAsubstitutions that diminish IL-2 production (which was seen with the MutH3.3 peptide) tell us critical AA for recognition by the TCR. Bothbinding—(FIG. 8A) and function (i.e., IL-2)—(FIG. 8B) based approachesconsistently showed that AA at positions 1, 2, 4, 6, 7, 8, 9 and 10 arecritical for the recognition. We then carried out an in silico search toidentify naturally existing AA sequences using NCBI BLAST. We found noknown human proteins that contain the same AA residues, indicating thatit would be highly unlikely that immunotherapy targeting this epitopewill cause unwanted off-target reactions against normal cells.

We also evaluated whether the H3.3.K27M-specific TCR is reactive to thedeiminated H3.3K.27M epitope using a synthetic peptide in which thearginine residue of the H3.3.K27M epitope is replaced by citrulline (CitH3.3) (FIG. 8A-B). While the Cit H3.3 peptide partially retains itsaffinity to HLA-A*0201 (FIG. 8A), it completely abrogates IL-2production by TCR-transduced J.RT3-T3.5 cells, indicating that the TCRdoes not recognize the Cit H3.3 peptide.

A subpopulation of glioma patients bears the K27M mutation in H3.1 (ahomologue of H3.3). The AA sequences encompassing the K27M mutation inH3.1 and H3.3 are similar. When compared with the H3.3.K27M epitope, thecorresponding portion of H.3.1 has only one AA substitution. Hence, weevaluated whether the TCR against the H3.3 K27M cross-reacts againstH3.1 K27M using a synthetic peptide designed for the putative H3.1 K27Mepitope (Mut H3.1 in FIG. 8B). The TCR failed to recognize the Mut H.3.1epitope, suggesting that patients with the H3.1.K27M mutation butwithout H3.3.K27M mutation are not eligible for prospectiveTCR-transduced adoptive transfer therapy.

Example 2

The H3.3K27M Peptide is Presented as an HLA-A*02:01+-Binding Epitope byGlioma Cells Harboring the H3.3K27M Mutation

To investigate whether the bioinformatically predicted H3.3K27M26-35peptide is produced and presented by the HLA machinery in HLA-A*02:01⁺H3.3K27M⁺ glioma cells, we used a recently developed mass spectrometry(MS)-based method for the direct identification of HLA-class I-bindingpeptides (17). We purified HLA-class I-binding peptides fromHLA-A*02:01⁺ U87MG glioma cells stably transduced with cDNA encodingH3.3K27M (U87H3.3K27M) or H3.3WT (U87H3.3WT), or parental U87MG cells,and analyzed them by LC-MS/MS using a Quadrupole Orbitrap MassSpectrometer. To specifically identify the H3.3K27M₂₆₋₃₅ peptide, weadded a fixed amount of a synthetic version of the H3.3K27M₂₆₋₃₅ 10-merto each sample containing a substitution of arginine at position 1 withthe heavy counterpart (¹³C6 ¹⁵N4 arginine), thus introducing a 10.0083Da mass difference. With this approach, the heavy labeled peptide wasreadily sequenced and identified because of its higher abundance duringLC-MS/MS analysis. Since both the heavy and light peptides elute at thesame time from the chromatographic column and differ exclusively in theintroduced mass change, it was possible to confidently identify theendogenous peptide even at extremely low concentrations and in theabsence of an MS/MS spectrum. Following re-calibration in MaxQuant, wedetected two co-eluting isotope patterns with mass to charge (m/z)ratios of 494.7420 and 489.7394 at 27.74 minutes of chromatographicseparation in U87H3.3K27M glioma cells (FIG. 9A). This was compatiblewith the predicted values for both the heavy and the endogenousH3.3K27M₂₆₋₃₅ peptides in their oxidized forms (494.7414 and 489.7373,respectively) with a relative mass difference of 10.0052 Da. The elutionpeak corresponding to the heavy peptide was selected multiple times forsequencing and its fragmentation spectrum was unequivocally identifiedas the oxidized form of the heavy H3.3K27M (Andromeda identificationscore 124.23; FIG. 9B). The putative light isotope pattern presentedcorrect m/z values for each of its isotopic peaks (FIG. 9C). Moreover,this isotope pattern was detectable exclusively in U87H3.3K27M cells butnot in either the parental U87 or the U87H3.3WT cells (data not shown).The modification observed in both peptides is due to the oxidation ofthe methionine residue during the experimental sample preparationprocedure and therefore does not reflect the state of the peptide invivo (18). Together, these observations demonstrate that theH3.3K27M₂₆₋₃₅ epitope peptide is naturally produced and presented by HLAclass I on the surface of glioma cells bearing the H3.3K27M mutation.

Human T-Cells Transduced with Retroviral Vector EncodingH3.3K27M-Specific TCR Demonstrate H3.3K27M-Specific CTL Reactivity

We isolated full-length cDNA for α- and β-chains of TCR from the clone1H5, optimized the codon usage, and cloned them into a TCR retroviralvector system, which incorporates small interfering RNA (siRNA)targeting constant regions of the endogenous TCR α- and β-chains toavoid mispairing between endogenous and transgene TCR chains (S.Okamoto, Y. Amaishi, Y. Goto, H. Ikeda, H. Fujiwara, K. Kuzushima, M.Yasukawa, H. Shiku, J. Mineno, Mol Ther Nucleic Acids 1, e63 (2012); S.Okamoto, J. Mineno, H. Ikeda, H. Fujiwara, M. Yasukawa, H. Shiku, I.Kato, Cancer Res 69, 9003-9011 (2009)) (FIG. 10A). To evaluate thefunction of the transgene TCR, we first transduced the Jurkat T-cellclone 76 (J76CD8) (21), which is deficient in endogenous TCR α- andβ-chains but expresses human CD8, with the TCR vector or with a mockvector. This experiment demonstrated that TCR-transduced but not controlmock-transduced J76CD8 cells produced elevated levels of IL-2 whenstimulated with the H3.3K27M peptide (FIG. 10B). Next, we transducedprimary human T-cells with the TCR and achieved over 40% transductionefficiency in CD8⁺ T-cells and approximately 20% in CD8⁻ T-cells basedon tetramer-staining, suggesting the CD8⁺ co-receptor is important forefficient expression of the TCR (FIG. 10C). We also observed asignificant up-regulation of the T-cell activation marker, CD69 (22), onTCR-transduced T-cells but not mock-transduced control T-cells, uponstimulation with the H3.3K27M26-35 mutant peptide but not WT or anadditional irrelevant, influenza-A-derived HLA-A*0201-binding peptide(FIG. 10D).

TCR-Transduced Primary T-Cells Recognize the H3.3K27M peptide at 10⁻⁸ to10⁻⁹ M

To evaluate the functional avidity of the TCR, we determined the peptideconcentration required to induce half-maximal response (EC₅₀), usingIFN-γ production by TCR-transduced T-cells as the readout of response.Using CD8⁺ T-cells derived from 3 healthy HLA-A*02:01⁺ donors andco-culturing them with T2 cells loaded with titrating doses of theH3.3K27M peptide, we determined the EC₅₀ of TCR-transduced T-cells to bebetween 10⁻⁸ to 10⁻⁹ M based on IFN-γ enzyme-linked immuno-spot(ELISPOT) assay (FIG. 11).

TCR-Transduced T-Cells Specifically Lyse H3.3K27M⁺HLA-A2⁺ Glioma CellsIn Vitro

It is essential to demonstrate that TCR-transduced T-cells are able torecognize the H3.3K27M epitope which is endogenously expressed in gliomacells, and lyse H3.3K27M⁺HLA-A*02:01⁺ glioma cells. To this end, weevaluated cytotoxicity of TCR-transduced T-cells againstH3.3K27M⁺HLA-A2⁺ HSJD-DIPG-017 cells using lactate dehydrogenase (LDH)detection-based cytotoxicity assay. While both TCR-transduced andcontrol mock-transduced T-cells were similarly activated, onlyTCR-transduced T-cells lysed HSJD-DIPG-017 cells. Addition of syntheticH3.3K27M peptide further enhanced the lysis by TCR-transduced T-cells(FIG. 12A). Furthermore, the observed lysis was dependent on HLA-A*02:01as TCR-transduced T-cells were not able to lyse HLA-A*02:01^(−neg)H3.3K27M⁺ HSJD-DIPG-019 cells, even in the presence of exogenously addedH3.3K27M peptide (FIG. 12A). To demonstrate that the reactivity ofTCR-transduced T-cells is specific to the H3.3K27M, we used HLA-A*02:01+U87MG glioma cells stably transduced with cDNA encoding H3.3K27M(U87H3.3K27M) or H3.3WT (U87H3.3WT; FIGS. 12B and C). TCR-transducedT-cells efficiently lysed U87H3.3K27M cells but not U87H3.3WT cells.Lysis of U87H3.3K27M cells was enhanced when synthetic H3.3K27M peptidewas added but abrogated by the anti-HLA-A2 blocking antibody. On theother hand, TCR-transduced T-cells lysed U87H3.3WT cells only whenloaded with synthetic H3.3K27M peptide (FIG. 12B). As an additionalmethod to confirm the H3.3K27M- and HLA-A*02:01-specific cytotoxicity ofTCR-transduced T-cells, we employed Carboxyfluorescein succinimidylester (CFSE)-stained target cells (U87H3.3K27M and U87H3.3WT cells) inthe co-culture assay and evaluated the expression of 7-aminoactinomycinD (7-AAD) on the CFSE⁺ target cells as an early marker for cell death(24). Our results confirmed those obtained by LDH-based assay (FIG.12C). These results indicate that TCR-transduced T-cells are able torecognize the H3.3K27M epitope that is processed and presented byH3.3K27M⁺HLA-A*02:01⁺ glioma cells and specifically lyse those gliomacells.

TCR-Transduced T-Cells Significantly Inhibit Progression of H3.3K27M⁺Glioma Xenograft

To determine the preclinical therapeutic activities of TCR-transducedT-cells in vivo, we injected 5×10⁴ U87H3.3K27M luciferase⁺ cells intothe brain of immunocompromised NSG mice on Day 0. On Day 14 and Day 30,mice with established tumors received intravenous injection of eitherPBS, 5×10⁶ control mock-transduced T-cells, or 5×10⁶ TCR-transducedT-cells. Bioluminescence imaging (BLI) indicated a significant reductionin the tumor burden in mice receiving TCR-transduced T-cells but not inmice receiving PBS or mock-transduced T-cells (FIGS. 13A and B). In anattempt to determine the accumulation of TCR-transduced T-cells in theintracranial xenograft, we euthanized the mice on Day 32 and evaluatedtumor-infiltrating lymphocytes using the HLA-A*02:01-H3.3K27M₂₆₋₃₅dextramer as well as anti-CD8 and anti-CD4 monoclonal antibodies. Whilethe transduction efficiency of the infused CD8⁺ and CD4⁺ cells wasapproximately 50% and 30%, respectively (FIG. 13C), we found that over80% and 40% of human CD8⁺ and CD4⁺ T-cells, respectively, were positivefor the dextramer in the tumor. On the other hand, approximately 10% and20% of CD8⁺ and CD4⁺ T-cells, respectively, were dextramer positive inthe peripheral blood (FIG. 13C). These data suggest that there was aselective accumulation of TCR-transduced T-cells in the intracranialtumor site.

Materials and Methods

Cells and Cell Culture.

DIPG neurosphere cultures were maintained in tumor stem media containingNeurobasal-A Medium (1×), DMEM/F-12, HEPES buffer, Sodium pyruvate, MEMnon-essential amino acids, GlutaMAX-I, Anti-Anti solution, and B-27supplement minus vitamin A or Gem21 neuroplex, all purchased from LifeTechnologies. Media was supplemented with 20 ng/mL recombinant human(rh) EGF (Peprotech; AF-100-15), 20 ng/mL rhFGF-basic (Peprotech;AF-100-18B), 10 ng/mL hPDGF-AA (Peprotech; 100-13A), 10 ng/mL hPDGF-BB(Peprotech; 100-14B) and 2 μg/ml Heparin solution (Sigma; H3149-10KU) atinitiation of cell culture and weekly thereafter. Cells were passaged bytrituration in TrypLE Express (Invitrogen; 12604-039) and DNase1(Worthington; LS002007) followed by resuspension in fresh media.U87H3.3K27M and U87H3.3WT were generated by transfection of a PT2/Cvector encoding cDNA for either WT H3.3 or the K27M H3.3 into parentalU87MG cells using Fugene HD transfection reagent (Promega; E2311).Expression of H3.3K27M was confirmed by western blot analysis with ananti-H3.3K27M antibody (Millipore; ABE419). The Jurkat T-cell clone 76(J76CD8) line (M. H. Heemskerk, M. Hoogeboom, R. A. de Paus, M. G.Kester, M. A. van der Hoorn, E. Goulmy, R. Willemze, J. H. Falkenburg,Blood 102, 3530-3540 (2003)), which are deficient in endogenous TCR α-and β-chains but express human CD8, was provided by Dr. Mirjam Heemskerk(Leiden University Medical Center, Leiden, Netherlands).

T-Cell Isolation.

LRS chambers containing healthy donor-derived HLA-A*02:01⁺ PBMCs wereobtained from the Stanford Blood Bank (Stanford, Calif.).Patient-derived PBMCs were obtained through the IRB-approvedNeurosurgery Tissue Bank (IRB/CHR #10-01318; PI Dr. Joanna Phillips)with coded tissue information without any protected health identifiers.T-cells were enriched from whole blood by immunodensity isolation usingthe RosetteSep™ Human T-cell Enrichment Cocktail (Stemcell Technologies;15061) according to the manufacturer's suggested protocol. T-cells werecryopreserved in RPMI media containing 20% human AB serum and 10% DMSOand stored at −196° C.

Peptides.

The synthetic peptides H3.3K27M₂₆₋₃₅ (RMSAPSTGGV (SEQ ID NO:2)),H3.3WT₂₆₋₃₅ (RKSAPSTGGV (SEQ ID NO:3)), CITmH3.3₂₆₋₃₅ (XMSAPSTGGV (SEQID NO:18)), H3.1K27M₂₆₋₃₅ (RMSAPATGGV (SEQ ID NO:6)), CEF1₅₈₋₆₆Influenza Matrix Protein M1 (GILGFVFTL (SEQ ID NO:19)), as well aspeptides used for the alanine scanning assay were synthesized by A&Alabs (San Diego, Calif.) and were >95% pure as indicated by analytichigh-performance liquid chromatography and mass spectrometric analysis.Peptides were dissolved in DMSO at a concentration of 10 mg/mL andstored at −80° C. until use.

HLA-Peptide Binding Assays.

Quantitative assays to measure the binding of peptides to purified HLAA*02:01 class I molecules are based on the inhibition of binding of aradiolabeled standard peptide (HBV core 18-27 analog, FLPSDYFPSV (SEQ IDNO:20)), and were performed as detailed elsewhere (J. Sidney, S.Southwood, C. Moore, C. Oseroff, C. Pinilla, H. M. Grey, A. Sette, CurrProtoc Immunol Chapter 18, Unit 18 13 (2013)). HLA molecules werepurified by affinity chromatography from the EBV transformed homozygouscell line JY, as described previously (J. Sidney, S. Southwood, C.Moore, C. Oseroff, C. Pinilla, H. M. Grey, A. Sette, Curr Protoc ImmunolChapter 18, Unit 18 13 (2013)). Peptides were tested at six differentconcentrations covering a 100,000-fold dose range in three or moreindependent assays, and the concentration of peptide yielding 50%inhibition of the binding of the radiolabeled probe peptide (IC₅₀) wascalculated. Under the conditions used, where [radiolabeled probe]<[MHC]and IC₅₀≥[MHC], the measured IC₅₀ values are reasonable approximationsof the true K_(d) values (K. Gulukota, J. Sidney, A. Sette, C. DeLisi, JMol Biol 267, 1258-1267 (1997); Y. Cheng, W. H. Prusoff, BiochemicalPharmacology 22, 3099-3108 (1973)).

ELISPOT Assays.

Patient-derived PBMCs were stimulated with 10 μg/ml H3.3K27M₂₆₋₃₅peptide or H3.3WT₂₆₋₃₅ peptide, or without peptide. At 48 hours, rhIL-2(50 U/ml), IL-7 (10 ng/ml) and IL-15 (10 ng/ml) were added to theculture for an additional 5 days. Fifty thousand peptide stimulatedT-cells were co-cultured with 5×10³ T2 cells pulsed with 10 μg/mlH3.3K27M₂₆₋₃₅ peptide, H3.3WT₂₆₋₃₅ peptide, or without peptide for 24hrs on the anti-human IFN-γ-antibody-coated ELISPOT plates. To determineTCR avidity, 5×10⁴ TCR-transduced CD8⁺ T-cells were co-cultured with5×10⁴ T2 cells pulsed with different concentrations of the H3.3K27Mpeptide overnight on the anti-human IFN-γ antibody-coated ELISPOTplates. The rest of the protocol was carried out according to themanufacturer's protocol (Human IFN-γ ELISPOT kit, BD, 552138). The spotswere quantified using the CTL S6 Universal-V Analyzer ELISpot Reader(ImmunoSpot®).

Purification and LC-MS/MS Analysis of HLA-Class I Peptides.

HLA-class I complexes were purified from 5×10⁸ U87 parental cells or U87cells transfected with either WT H3.3 or H3.3K27M, as previouslydescribed (17). Briefly, cells were lysed with 0.25% sodium deoxycholate(Sigma-Aldrich), 1% octyl-β-D glucopyranoside (Sigma-Aldrich), 0.2 mMiodoacetamide, 1 mM EDTA, and 1:200 Protease Inhibitors Cocktail(Sigma-Aldrich) in PBS at 4° C. for 1 hour. The lysate was cleared by a30 minute centrifugation at 40,000×g at 4° C. HLA-class I complexes wereimmunoaffinity-purified from the cleared lysate with Protein-A Sepharosebeads (Invitrogen) covalently bound to the pan-HLA-class I antibodyW6/32 (purified from HB95 cells, ATCC) and eluted at room temperaturewith 0.1 N acetic acid. Eluted HLA-I complexes were then loaded onSep-Pak tC18 cartridges (Waters) and HLA-class I peptides were separatedfrom the complexes by eluting them with 30% acetonitrile (ACN) in 0.1%trifluoroacetic acid (TFA). Peptides were further purified using SilicaC-18 column tips (Harvard Apparatus), eluted again with 30% ACN in 0.1%TFA and concentrated by vacuum centrifugation. For LC-MS/MS analysis,HLA-class I peptides were separated on an EASY-nLC 1000 system (ThermoFisher Scientific) coupled on-line to a Q Exactive HF mass spectrometer(Thermo Fisher Scientific) with a nanoelectrospray ion source (ThermoFisher Scientific). Peptides were loaded in buffer A (0.1% formic acid)into a 50 cm long, 75 μm inner diameter column in house packed withReproSil-Pur C18-AQ 1.9 μm resin (Dr. Maisch HPLC GmbH) and eluted witha 90 minute linear gradient of 5-30% buffer B (80% ACN, 0.1% formicacid) at a 250 nl/min flow rate. The Q Executive HF operated in a datadependent mode with full MS scans at the range of 300-1,650 m/z, withresolution of 60,000 at 200 m/z and the target value of 3×10⁶ ions. Theten most abundant ions with charge between 1 and 3 were combined to givean AGC target value of 1×10⁵ and for a maximum injection time of 120 msand fragmented by higher-energy collisional dissociation (HCD). MS/MSscans were acquired with a resolution of 15,000 at 200 m/z and dynamicexclusion was set to 20s in order to avoid repeated peptide sequencing.Data were acquired and analyzed with the Xcalibur software (ThermoScientific). For the targeted identification of the H3.3K27M peptide, aheavy (Arg10) version of the peptide was synthesized with the Fmoc solidphase method using the ResPepMicroScale instrument (Intavis AGBioanalytical instruments), introducing a 10.0083 Da mass differencewith one ¹³C6 ¹⁵N4 arginine (AnaSpec Inc.). The heavy peptide was addedto each sample at a 1 pmol/μl concentration for a total of 3 pmoles perrun.

For mass spectrometry data analysis, raw files were processed usingMaxQuant version 1.5.7.12 as described previously (M. Bassani-Sternberg,S. Pletscher-Frankild, L. J. Jensen, M. Mann, Mol Cell Proteomics 14,658-673 (2015)). Searches were performed against the Human UniProtdatabase (July 2015) and a customized reference database containing theH3.3K27M sequence. Enzyme specificity was set as unspecific and possiblesequences matches were restricted to 8-15 amino acids and a maximum massof 1500 Da. N-terminal acetylation and methionine oxidation were set asvariable modifications. A false discovery rate of 0.01 was required atthe peptide level. To identify the heavy peptide, an Arg10 label wasadded to the search type specification.

Induction and Isolation of H3.3K27M-Specific CTL Clones.

To generate dendritic cells, the plastic adherent cells from PBMCs werecultured in AIM-V medium (Invitrogen) supplemented with 1,000 units/mLrecombinant human granulocyte macrophage colony-stimulating factor and500 units/mL recombinant human IL-4 (rhIL-4; Cell Sciences) at 37° C. ina humidified CO₂ (5%) incubator. Six days later, the immature dendriticcells were stimulated with recombinant human tumor necrosis factor-α,IL-6, and IL-1β (10 ng/mL each). Mature dendritic cells were thenharvested on day 8, resuspended in AIM-V medium at 1×10⁶ cells per mLwith peptide (10 μg/mL), and incubated for 2 hours at 37° C. Populationsof autologous CD8⁺ T-cells were enriched from PBMCs using magneticmicrobeads (Miltenyi Biotech). CD8⁺ T-cells (2×10⁶ per well) wereco-cultured with 2×10⁵ per well peptide-pulsed dendritic cells in 2mL/well of AIM-V medium supplemented with 5% human AB serum, 10 units/mLrhIL-2 (R&D Systems, Minneapolis, Minn.), and 10 units/mL rhIL-7 (CellSciences) in each well of 24-well tissue culture plates. On day 15,lymphocytes were re-stimulated with autologous dendritic cells pulsedwith peptide in AIM-V medium supplemented with 5% human AB serum,rhIL-2, and rhIL-7 (10 units/mL each).

HLA-A*0201-Peptide Tetramer Staining.

Phycoerythrin (PE)-conjugated HLA-A*0201 RMSAPSTGGV (SEQ ID NO:2)tetramer (H3.3K27M-tetramer) was produced by the National Institute ofAllergy and Infectious Disease tetramer facility within the EmoryUniversity Vaccine Center (Atlanta, Ga.) using the peptide synthesizedby A&A labs (San Diego, Calif.). PE-conjugated HLA-A*0201/RMSAPSTGGV(SEQ ID NO:2) Dextramer was purchased from Immudex (Denmark). Cells werestained with tetramer (10 μg/mL) or Dextramer in PBS containing 1%bovine serum albumin (FACS Buffer) for 15 minutes at 4° C. (fortetramer) or room temperature (for dextramer), followed by surfacestaining for various T-cell markers at 4° C. Cells were then washed withFACS Buffer (PBS containing 1% FBS). For some experiments, T-cells werestained with tetramer, followed by anti-human CD8 APC (Biolegend,344722) and anti-human CD69 FITC (eBioscience, 11-0699-42).

ELISA.

Media was collected and centrifuged at 500 g for 10 minutes to removedebris. The human IFN-γ (BD OptEIA, 555142) and human IL-2 (ThermoFischer Scientific, EH2IL2), ELISA was carried out according to themanufacturer's protocol. Plates were analyzed on a Biotek Synergy2microplate reader (Biotek) at wavelengths of 450 nm and a background of550 nm.

Cloning of TCR.

5′-Rapid Amplification of cDNA Ends-PCR (RACE-PCR) was performed bySMARTer® RACE 5′/3′ Kit (Clontech Laboratories; 634838). Briefly, SingleRACE PCR product (˜900 bp) was excised from a 1.2% agarose gel, purifiedby Zymoclean™ Gel DNA Recovery Kit (Zymo; D4001) and TOPO® cloned intopCR™-Blunt II-TOPO® using the Zero Blunt® TOPO® PCR Cloning Kit (LifeTechnologies; K2800-20SC). The cloned product was then transformed intoMAX Efficiency® DH5α™ Competent cells (Life Technologies; 182580120) andplated on ampicillin-containing LB agar plates (TEKNOVA; L1004). LB agarplates were placed at 37° C. overnight to allow colonies to form.Selected clones from the resulting constructs were subjected to PCRamplification and sequencing (QuintaraBio; San Francisco, Calif.) byusing the M13 (−21) forward primer (5′ TGTAAAACGACGGCCAGT-3′ (SEQ IDNO:21)) and M13 reverse primer (5′-CAGGAAACAGCTATGAC-3′ (SEQ ID NO:22))for sequencing. Sequences were analyzed using SnapGene Viewer (SnapGene, Chicago, Ill.).

Generation of siTCR Retroviral Producer Cell Lines.

The siTCR system has been previously described (S. Okamoto, Y. Amaishi,Y. Goto, H. Ikeda, H. Fujiwara, K. Kuzushima, M. Yasukawa, H. Shiku, J.Mineno, Mol Ther Nucleic Acids 1, e63 (2012).; S. Okamoto, J. Mineno, H.Ikeda, H. Fujiwara, M. Yasukawa, H. Shiku, I. Kato, Cancer Res 69,9003-9011 (2009)). Briefly, codon optimized TCRA and TCRB fragments wereartificially synthesized and cloned into Takara's siTCR retroviralvector plasmid. PG13 packaging cells were plated at 3×10⁴ cells per wellof a six-well plate and incubated for 24 hours. PG13 cells weretransfected with siTCR retroviral vector in the presence of 8 μg/mL ofpolybrene, and incubated for 4 hours at 37° C., 5% CO₂, this wasrepeated on the following day. The cells were further expanded and werethen harvested as GaLV envelope pseudo-typed siTCR retroviral vectorproducer cells and were cryopreserved. For generation of retroviralparticles, culture supernatant was collected from cells grown incomplete DMEM media supplemented with 5 mM sodium butyrate(Sigma-Aldrich, 156-54-7) for 24 hours.

Infection of Primary T-Cells with siTCR Vector.

Human PBMCs were activated on plates pre-coated with anti-human CD3antibody (OKT3 clone, Miltenyi Biotec, 170-076-124) and RetroNectin®(RN, Takara Bio, T100A). Three days after the stimulation, viralsupernatant was spun on the RetroNectin coated plates at 2,000 g for 2hrs at 4° C. Activated PBMCs were then added to the virus-coatedRetroNectin plates using spinfection methodology at 1,000 g for 10 minsat 4° C., and the cells were supplemented with 600 U/ml IL-2 (Peprotech,200-02). This transduction protocol was repeated on the next day, andPBMCs were allowed to rest for an additional 4 days and then stainedwith HLA-A*02:01-H3.3K27M tetramer to determine the transductionefficiency. The T-cells were maintained in 100 U/ml rhIL-2-containingfreshly made GT-T551 media (Takara Bio, WK551S).

LDH-Based Cytotoxicity Assay.

The CytoTox 96 non-radioactive cytotoxicity assay (Promega) was carriedout according to the manufacturer's protocol. Target cells were platedin 96 well plates with various Effector to Target ratios in 200 μl mediafor 24 hours. Fifty μl of supernatant was then transferred to anenzymatic assay plate containing 50 μl of CytoTox 96 Reagent andincubated for 30 minutes at room temperature. Stop solution was thenadded to each well and plates were analyzed at 490 nm on a Synergy2microplate reader (Biotek).Percent cytotoxicity was calculated as [(Experimental−Effectorspontaneous−Target Spontaneous)/(Target Maximum−TargetSpontaneous)]×100.

CSFE-Based Cytotoxicity Assay.

Target cells were stained with carboxyfluorescein succinimidyl ester(CFSE) using the Vybrant® CFDA SE Cell Tracer Kit (Thermo FisherScientific, V12883). CFSE-labelled target cells (5×10⁴/well) wereincubated with CTLs at the E/T ratio of 5 for 8 hours. To blockHLA-A2-mediated lysis, anti-HLA-A2 antibody (10 μg/ml, Biolegend,343302) was added to one group per experiment. At the end of incubation,7-AAD (Biolegend, 420403) was added into each well and incubated for 10minutes on ice. The samples were analyzed by flow cytometry, and thekilled target cells were identified as CFSE⁺7-AAD⁺ cells. Thecytotoxicity was calculated as the percentage of CFSE⁺ and 7-AAD⁺ cellsin total CFSE cells.

Therapy of Mice Bearing Intracranial Glioma Xenografts.

Five- to 6-week-old NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wjl)/SzJ (NSG mice)female mice (Jackson Laboratory, Bar Harbor, Me.) were used in theexperiments. Animals were handled in the Animal Facility at theUniversity of California, San Francisco per an Institutional Animal Careand Use Committee-approved protocol. The procedure has been previouslydescribed by us (M. Ohno, T. Ohkuri, A. Kosaka, K. Tanahashi, C. H.June, A. Natsume, H. Okada, Journal for Immunotherapy of Cancer 1, 21(2013)). Briefly, using a stereotactic apparatus, mice received 5×10⁴U87H3.3K27M cells/mouse in 2 μl PBS at 2 mm lateral to the bregma and 3mm below the surface of the skull. After tumors were established, eachmouse received intravenous infusion of PBS, mock-transduced T-cells or5×10⁶ TCR-transduced via the tail vein on Days 10 and 30 post tumorinoculation.

Bioluminescence Imaging.

The growth of luciferase positive U87H3.3K27M tumors in the brain wasnon-invasively monitored by BLI using the in vivo imaging system IVIS100 (PerkinElmer, Alameda, Calif.). Mice received intraperitonealinjection with 200 μl (15 mg/ml) of freshly thawed aqueous solution ofD-Luciferin potassium salt (PerkinElmer), were anesthetized withisoflurane, and imaged for bioluminescence for 1 min exposure time.Optical images were analyzed by IVIS Living Image software package.

Statistical Analyses.

All statistical analyses were carried out on Graphpad Prism software.For in vitro studies, Student t-test or one-way ANOVA were used tocompare two groups or more than two groups, respectively. Non-linearregression analysis was used to determine the EC₅₀. We considereddifferences significant when p<0.05.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A modified T-cell comprising a heterologousT-cell receptor (TCR), or fragment thereof, which binds to apeptide/major histocompatibility complex (MHC) complex, wherein the TCRor the fragment thereof binds to a histone H3 variant H3.3 peptide,wherein the TCR or fragment thereof comprises: a TCR alpha chain orfragment thereof comprising complementarity determining regions (CDRs)1, 2, and 3 comprising SEQ ID NOs: 12, 13, and 14, respectively; and aTCR beta chain or fragment thereof comprising CDRs 1, 2, and 3comprising SEQ ID NOs: 15, 16, and 17, respectively.
 2. The T-cell ofclaim 1, wherein the TCR alpha chain or fragment thereof is a TCR alphachain and the TCR beta chain or fragment thereof is a TCR beta chain. 3.The T-cell of claim 2, wherein the TCR alpha chain comprises the aminoacid sequence set forth in SEQ ID NO: 8, and the TCR beta chaincomprises the amino acid sequence set forth in SEQ ID NO:
 10. 4. TheT-cell of claim 3, wherein the heterologous TCR is expressed from anucleic acid comprising a polynucleotide sequence encoding aself-cleaving peptide that links polynucleotide sequences encoding theTCR alpha and beta chains.
 5. The T-cell of claim 4, wherein theself-cleaving peptide is a porcine teschovirus-1 2A (P2A) peptide. 6.The T-cell of claim 1, wherein the heterologous TCR is expressed from aretroviral vector.
 7. The T-cell of claim 6, wherein the retroviralvector is a lentiviral vector.
 8. The T-cell of claim 7, wherein theT-cell exhibits downregulated expression of an endogenous T-cellreceptor comprising an endogenous TCR alpha chain and an endogenous TCRbeta chain.
 9. The T-cell of claim 8, wherein i) the T-cell exhibitsdownregulated expression of an endogenous TCR alpha chain; ii) theT-cell exhibits downregulated expression of an endogenous TCR betachain; or iii) the T-cell exhibits downregulated expression of anendogenous TCR alpha chain and downregulated expression of an endogenousTCR beta chain.
 10. The T-cell of claim 9, wherein i) the T-cellexhibits downregulated expression of an endogenous TCR alpha chain dueto expression of an siRNA complementary to the endogenous TCR alphachain; ii) the T-cell exhibits downregulated expression of an endogenousTCR beta chain due to expression of an siRNA complementary to theendogenous TCR beta chain; or iii) the T-cell exhibits downregulatedexpression of an endogenous TCR alpha chain due to expression of ansiRNA complementary to the endogenous TCR alpha chain and downregulatedexpression of an endogenous TCR beta chain due to expression of an siRNAcomplementary to the endogenous TCR beta chain.
 11. The T-cell of claim10, wherein i) the lentiviral vector further expresses the siRNA capableof downregulating expression of an endogenous TCR alpha chain; ii) thelentiviral vector further expresses the siRNA capable of downregulatingexpression of an endogenous TCR beta chain; or iii) the lentiviralvector further expresses the siRNA capable of downregulating expressionof an endogenous TCR alpha chain and the siRNA capable of downregulatingexpression of an endogenous TCR beta chain.
 12. The T-cell of claim 1,wherein expression of the heterologous TCR is under the control of aheterologous promoter.
 13. The T-cell of claim 12, wherein theheterologous promoter is a constitutive promoter.
 14. The T-cell ofclaim 11, wherein expression of the siRNA capable of downregulatingexpression of an endogenous TCR alpha chain and expression of the siRNAcapable of downregulating expression of an endogenous TCR beta chain areunder the control of a heterologous promoter.
 15. The T-cell of claim 1,wherein the peptide is in a complex with a MHC.
 16. The T-cell of claim1, wherein the peptide in the peptide/WIC complex comprises the aminoacid sequence (R/A)MSAP(S/A)TGGV (SEQ ID NO: 1).
 17. The T-cell of claim16, wherein the peptide in the peptide/MHC complex consists of 10-12amino acids.
 18. The T-cell of claim 17, wherein the amino acid sequenceis RMSAPSTGGV (SEQ ID NO: 2).